Odgren Lab Copyright (c) 2013 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/odgren Recent documents in Odgren Lab en-us Wed, 13 Feb 2013 17:54:05 PST 3600 Molecular cloning and characterization of rat CCL9 (MIP-1gamma), the ortholog of mouse CCL9 http://escholarship.umassmed.edu/odgren/13 http://escholarship.umassmed.edu/odgren/13 Wed, 16 Feb 2011 09:07:41 PST We identified an EST sequence that was up-regulated during osteoclast formation in the rat. Investigating further, we cloned the cDNA from rat long bone and found it to be highly homologous to the mouse CC chemokine, CCL9, both at the nucleotide and amino acid levels. The rat CCL9 amino acid sequence is 74% identical to the mouse sequence, with an additional 11% similar amino acids. Recombinant rat CCL9 was used in chemotaxis assays of rat bone marrow cells and it was found to have a strong and dose-dependent effect. In addition, CCL9 mRNA was very highly up-regulated during osteoclast differentiation of rat bone marrow-derived mononuclear cells, increasing by over 100-fold when stimulated by colony stimulating factor-1 and the TNF superfamily member, RANKL. Together, these results establish that, like the mouse, the rat also uses CCL9 to promote the complex process of osteoclast formation.

]]> Meilheng Yang et al. Amino Acid Sequence Animals Base Sequence Chemokines, CC Chemotaxis Cloning, Molecular DNA, Complementary Macrophage Inflammatory Proteins Mice Molecular Sequence Data Osteoclasts Polymerase Chain Reaction RNA, Messenger Rats Sequence Homology, Amino Acid Sequence Homology, Nucleic Acid Species Specificity CSF-1 regulation of osteoclastogenesis for tooth eruption http://escholarship.umassmed.edu/odgren/12 http://escholarship.umassmed.edu/odgren/12 Wed, 16 Feb 2011 09:07:40 PST The dental follicle regulates the alveolar bone resorption needed for tooth eruption. In the rat first mandibular molar, a decrease in the expression of osteoprotegerin (OPG) in the dental follicle at day 3 enables the osteoclastogenesis needed for eruption to occur. Because colony-stimulating factor-1 (CSF-1) is maximally expressed in the dental follicle at day 3, it was hypothesized that CSF-1 down-regulates OPG gene expression in the dental follicle in vivo. To test this, we compared the expression of OPG in osteopetrotic toothless (tl/tl) rats deficient in CSF-1 with expression in their normal littermates for given ages. OPG gene expression was found to be higher in the dental follicle of the tl/tl mutants than in normals. Transfecting short interfering RNA specific for CSF-1 mRNA into dental follicle cells resulted in an up-regulation of OPG expression. Thus, these studies support our hypothesis that the down-regulation of OPG needed for tooth eruption is mediated by CSF-1.

]]> Gary E. Wise et al. Animals Dental Sac Down-Regulation Gene Expression Regulation, Developmental Glycoproteins Immunoenzyme Techniques Macrophage Colony-Stimulating Factor Osteoclasts Osteoprotegerin RNA, Small Interfering Rats Rats, Mutant Strains Receptors, Cytoplasmic and Nuclear Receptors, Tumor Necrosis Factor Reverse Transcriptase Polymerase Chain Reaction Tooth Eruption Transfection Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis http://escholarship.umassmed.edu/odgren/11 http://escholarship.umassmed.edu/odgren/11 Wed, 16 Feb 2011 09:07:39 PST Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

]]> Nacksung Kim et al. Animals Carrier Proteins Cell Differentiation DNA Primers Gene Deletion Hematopoietic Stem Cells Histological Techniques Lymphotoxin-alpha Male Membrane Glycoproteins Mice Mice, Inbred C57BL Mice, Knockout Osteoclasts RANK Ligand Receptor Activator of Nuclear Factor-kappa B Reverse Transcriptase Polymerase Chain Reaction Signal Transduction TNF Receptor-Associated Factor 6 Tumor Necrosis Factor-alpha In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method http://escholarship.umassmed.edu/odgren/10 http://escholarship.umassmed.edu/odgren/10 Wed, 16 Feb 2011 09:07:38 PST INTRODUCTION: Isolating and culturing primary chondrocytes such that they retain their cell type and differentiate to a hypertrophic state is central to many investigations of skeletal growth and its regulation. The ability to store frozen chondrocytes has additional scientific and tissue engineering interest. Previous work has produced approaches of varying yield and complexity but does not permit frozen storage of cells for subsequent differentiation in culture. Investigations of growth plate dysplasias secondary to defective osteoclastogenesis in rodent models of osteopetrosis led us to adapt and modify a culture method and to cryopreserve neonatal rat costochondral chondrocytes.

METHODS: Chondrocytes were isolated from dissected ribs of 3-day-old rat pups by collagenase, hyaluronidase, and trypsin serial digestions. This was done either immediately or after the isolation was interrupted following an initial protease treatment to allow the chondrocytes, still in partially digested rib rudiments, to be frozen and later thawed for culture. Cells were plated in flat-bottom wells and allowed to adhere and grow under different conditions. Choice of media permitted cells to be maintained or induced to differentiate. Cell growth was monitored, as was expression of several relevant genes: collagen types II and X; osteocalcin, Sox9, adipocyte FABP, MyoD, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were examined by light, fluorescence, and electron microscopy.

RESULTS: Cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix even after freezing-thawing. RT-PCR showed retention of chondrocyte-specific gene expression, including aggrecan and collagen II. The cells had a flattened, "proliferating zone" appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellular matrix, along with matrix vesicles. The switch to collagen type X as marker for hypertrophy was not rigidly temporally regulated as happens in vivo, but its expression increased during hypertrophic differentiation.

CONCLUSIONS: This method should prove valuable as a means of studying chondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to stockpile frozen raw material for subsequent studies.

]]> Alison Gartland et al. Animals Animals, Newborn Base Sequence *Cell Differentiation Cells, Cultured Chondrocytes Cryopreservation DNA Primers Gene Expression Immunohistochemistry Microscopy, Electron Rats Reverse Transcriptase Polymerase Chain Reaction False-positive beta-galactosidase staining in osteoclasts by endogenous enzyme: studies in neonatal and month-old wild-type mice http://escholarship.umassmed.edu/odgren/9 http://escholarship.umassmed.edu/odgren/9 Wed, 16 Feb 2011 09:07:37 PST Escherichia coli beta-galactosidase (beta-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by beta-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined beta-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). beta-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using beta-gal as a marker gene.

]]> Paul R. Odgren et al. Acid Phosphatase Animals Animals, Newborn Bone and Bones False Positive Reactions Histocytochemistry Isoenzymes Mice Mice, Inbred C57BL Osteoclasts beta-Galactosidase Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans http://escholarship.umassmed.edu/odgren/8 http://escholarship.umassmed.edu/odgren/8 Wed, 16 Feb 2011 09:07:36 PST This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

]]> Liesbeth van Wesenbeeck et al. Adaptor Proteins, Signal Transducing Adaptor Proteins, Vesicular Transport Animals Chromosome Mapping *Chromosomes, Human, Pair 10 Female Gene Expression Regulation Humans Kidney Male Membrane Glycoproteins Monocytes Mutation Organ Specificity Osteopetrosis Pedigree Rats rab GTP-Binding Proteins Temporal evolution of gene expression in rat carotid artery following balloon angioplasty http://escholarship.umassmed.edu/odgren/7 http://escholarship.umassmed.edu/odgren/7 Wed, 16 Feb 2011 09:07:35 PST The success of vascular intervention including angioplasty, stenting, and arterial bypass remains limited by negative remodeling resulted in lumen restenosis. This study was to characterize the global transcription profile reflecting concurrent events along arterial remodeling and neointima formation in a rat carotid artery balloon-injury model. Expression profiling of injured and control common carotid arteries on days 4, 7, 14 post-injury that mark the major pathohistological progression stages of neointimal formation were recorded on high-density oligonucleotide arrays. A subset of genes from microarray-based data was further studied using quantitative real time RT-PCR and in situ hybridization with sequential arterial samples from days 1 to 28 post-injury. The gene-encoded proteins were validated with Western blot. Besides temporal induction of a large cluster of genes over-represented by cell proliferation and macromolecule metabolism gene ontology categories, a fast-evolving inflammation could be demonstrated by the induction of Tgfb and other anti-inflammatory genes (e.g., C1qtnf3 (C1q and tumor necrosis factor related protein 3 (predicted))) and a shift from type 1 to 2 helper T cell response. The most significant signature of the induced neointimal profile is enrichment of genes functionally related to angiogenesis and extracellular matrix (ECM) remodeling (e.g., Spp1 (secreted phosphoprotein 1), CD44 (CD44 antigen), and Cxcl12 (chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)). Some of the genes represent stress-responsive mesenchymal stromal cell cytokines. This study highlighted mesenchymal stromal cell cytokines-driven inflammatory extracellular matrix remodeling, as target processes for potential clinical therapeutic intervention.

]]> Jian-ming Li et al. *Angioplasty, Balloon Animals *Carotid Arteries Computational Biology Gene Expression Profiling *Gene Expression Regulation Hyperplasia In Situ Hybridization Intercellular Signaling Peptides and Proteins Male Oligonucleotide Array Sequence Analysis Oxidative Stress Rats Rats, Sprague-Dawley NHA-oc/NHA2: a mitochondrial cation-proton antiporter selectively expressed in osteoclasts http://escholarship.umassmed.edu/odgren/6 http://escholarship.umassmed.edu/odgren/6 Wed, 16 Feb 2011 09:07:34 PST Bone resorption is regulated by a complex system of hormones and cytokines that cause osteoblasts/stromal cells and lymphocytes to produce factors including RANKL, that ultimately result in the differentiation and activation of osteoclasts, the bone resorbing cells. We used a microarray approach to identify genes upregulated in RANKL-stimulated osteoclast precursor cells. Osteoclast expression was confirmed by multiple tissue Northern and in situ hybridization analysis. Gene function studies were carried out by siRNA analysis. We identified a novel gene, which we termed nha-oc/NHA2, which is strongly upregulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation-proton antiporter (CPA) and is the mouse orthologue of a human gene identified in a database search: HsNHA2. nha-oc/NHA2 is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na(+)-dependent changes in mitochondrial pH and Na(+) acetate induced mitochondrial passive swelling. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and resorption, suggesting a role for NHA-oc/NHA2 in these processes. nha-oc/NHA2 therefore is a novel member of the CPA family and is the first mitochondrial NHA characterized to date. nha-oc/NHA2 is also unique in that it is the first eukaryotic and tissue-specific CPA2 characterized to date. NHA-oc/NHA2 displays the expected activities of a bona fide CPA and plays a key role(s) in normal osteoclast differentiation and function.

]]> Ricardo A. Battaglino et al. Amino Acid Sequence Animals Antiporters Caspases Cell Differentiation Cell Line Cloning, Molecular Enzyme Activation Gene Expression Regulation Humans Hydrogen-Ion Concentration Mice Mitochondria Mitochondrial Swelling Molecular Sequence Data Osteoclasts RNA, Messenger RNA, Small Interfering Refined genomic localization of the genetic lesion in the osteopetrosis (op) rat and exclusion of three positional and functional candidate genes, Clcn7, Atp6v0c, and Slc9a3r2 http://escholarship.umassmed.edu/odgren/4 http://escholarship.umassmed.edu/odgren/4 Wed, 16 Feb 2011 09:07:33 PST Osteopetrosis is a disease characterised by a generalized skeletal sclerosis resulting from a reduced osteoclast-mediated bone resorption. Several spontaneous mutations lead to osteopetrotic phenotypes in animals. Moutier et al. (1974) discovered the osteopetrosis (op) rat as a spontaneous, lethal, autosomal recessive mutant. op rats have large nonfunctioning osteoclasts and severe osteopetrosis. Dobbins et al. (2002) localized the disease-causing gene to a 1.5-cM genetic interval on rat chromosome 10, which we confirm in the present report. We also refined the genomic localization of the disease gene and provide statistical evidence for a disease-causing gene in a small region of rat chromosome 10. Three strong functional candidate genes are within the delineated region. Clcn7 was previously shown to underlie different forms of osteopetrosis, in both human and mice. ATP6v0c encodes a subunit of the vacuolar H(+)-ATPase or proton pump. Mutations in TCIRG1, another subunit of the proton pump, are known to cause a severe form of osteopetrosis. Given the critical role of proton pumping in bone resorption, the Slc9a3r2 gene, a sodium/hydrogen exchanger, was also considered as a candidate for the op mutation. RT-PCR showed that all 3 genes are expressed in osteoclasts, but sequencing found no mutations either in the coding regions or in intron splice junctions. Our ongoing mutation analysis of other genes in the candidate region will lead to the discovery of a novel osteopetrosis gene and further insights into osteoclast functioning.

]]> B. Perdu et al. Animals Bone and Bones Chloride Channels Chromosome Mapping Cytoskeletal Proteins Disease Models, Animal Exons Genetic Predisposition to Disease Introns Ion Pumps Male Mutation Osteoclasts Osteopetrosis Proton-Translocating ATPases Rats Rats, Inbred Lew Rats, Mutant Strains Vacuolar Proton-Translocating ATPases Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation http://escholarship.umassmed.edu/odgren/5 http://escholarship.umassmed.edu/odgren/5 Wed, 16 Feb 2011 09:07:33 PST Microarray and real-time RT-PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC-STAMP. Specific siRNA and antibodies were used to inhibit OC-STAMP RNA and protein, respectively, and tartrate-resistant acid phosphatase (TRAP)+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/- RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL-induced differentiation was studied. OC-STAMP was very strongly up-regulated during osteoclast differentiation. Northern blots and sequence analysis revealed two transcripts of 2 and 3.7 kb differing only in 3'UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multipass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy-terminal 193 amino acids, however, are significantly similar to the DC-STAMP family consensus sequence. DC-STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC-STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of TRAP+, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC-STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC-STAMP is a previously unknown, RANKL-induced, multipass transmembrane protein that promotes the formation of multinucleated osteoclasts.

]]> Meilheng Yang et al. Acid Phosphatase Amino Acid Sequence Animals Antibodies Bone Marrow Cells Bone and Bones Cell Differentiation Cell Nucleus Cells, Cultured Immunohistochemistry Isoenzymes Membrane Proteins Mice Mice, Knockout Microarray Analysis Molecular Sequence Data Osteoclasts RANK Ligand RNA, Messenger RNA, Small Interfering Rats Reverse Transcriptase Polymerase Chain Reaction Sequence Analysis Transfection Up-Regulation Septoclast deficiency accompanies postnatal growth plate chondrodysplasia in the toothless (tl) osteopetrotic, colony-stimulating factor-1 (CSF-1)-deficient rat and is partially responsive to CSF-1 injections http://escholarship.umassmed.edu/odgren/3 http://escholarship.umassmed.edu/odgren/3 Wed, 16 Feb 2011 09:07:32 PST The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1(tl)). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1(tl)/Csf1(tl) rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1(tl)/Csf1(tl) mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1(tl)/Csf1(tl) rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.

]]> Alison Gartland et al. Animals Bone Development Bone Diseases, Developmental Cartilage Chondrocytes Growth Plate Immunohistochemistry Macrophage Colony-Stimulating Factor Neovascularization, Physiologic Osteopetrosis Rats Rats, Mutant Strains Disheveled hair and ear (Dhe), a spontaneous mouse Lmna mutation modeling human laminopathies http://escholarship.umassmed.edu/odgren/2 http://escholarship.umassmed.edu/odgren/2 Wed, 16 Feb 2011 09:07:31 PST BACKGROUND: Investigations of naturally-occurring mutations in animal models provide important insights and valuable disease models. Lamins A and C, along with lamin B, are type V intermediate filament proteins which constitute the proteinaceous boundary of the nucleus. LMNA mutations in humans cause a wide range of phenotypes, collectively termed laminopathies. To identify the mutation and investigate the phenotype of a spontaneous, semi-dominant mutation that we have named Disheveled hair and ear (Dhe), which causes a sparse coat and small external ears in heterozygotes and lethality in homozygotes by postnatal day 10.

FINDINGS: Genetic mapping identified a point mutation in the Lmna gene, causing a single amino acid change, L52R, in the coiled coil rod domain of lamin A and C proteins. Cranial sutures in Dhe/+ mice failed to close. Gene expression for collagen types I and III in sutures was deficient. Skulls were small and disproportionate. Skeletons of Dhe/+ mice were hypomineralized and total body fat was deficient in males. In homozygotes, skin and oral mucosae were dysplastic and ulcerated. Nuclear morphometry of cultured cells revealed gene dose-dependent blebbing and wrinkling.

CONCLUSION: Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies.

]]> Paul R. Odgren et al. Lamins Mutation PAMM: a redox regulatory protein that modulates osteoclast differentiation http://escholarship.umassmed.edu/odgren/1 http://escholarship.umassmed.edu/odgren/1 Wed, 16 Feb 2011 09:07:30 PST The central role of reactive oxygen species (ROS) in osteoclast differentiation and in bone homeostasis prompted us to characterize the redox regulatory system of osteoclasts. In this report, we describe the expression and functional characterization of PAMM, a CXXC motif-containing peroxiredoxin 2-like protein expressed in bone marrow monocytes on stimulation with M-CSF and RANKL. Expression of wild-type (but not C to G mutants of the CXXC domain) PAMM in HEK293 cells results in an increased GSH/GSSG ratio, indicating a shift toward a more reduced environment. Expression of PAMM in RAW264.7 monocytes protected cells from hydrogen peroxide-induced oxidative stress, indicating that PAMM regulates cellular redox status. RANKL stimulation of RAW 264.7 cells caused a decrease in the GSH/GSSG ratio (reflecting a complementary increase in ROS). In addition, RANKL-induced osteoclast formation requires phosphorylation and translocation of NF-kappaB and c-Jun. In stably transfected RAW 264.7 cells, PAMM overexpression prevented the reduction of GSH/GSSG induced by RANKL. Concurrently, PAMM expression completely abolished RANKL-induced p100 NF-kappaB and c-Jun activation, as well as osteoclast formation. We conclude that PAMM is a redox regulatory protein that modulates osteoclast differentiation in vitro. PAMM expression may affect bone resorption in vivo and help to maintain bone mass.

]]> Yan Xu et al. Reactive Oxygen Species Oxidation-Reduction Osteoclasts Cell Differentiation