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ABSTRACT: A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. This article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order small inhibitory RNAs (siRNAs) designed to "knockdown" expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically and presented to the class in groups. In the past 2 years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.
Biochemistry and Molecular Biology Education 11/2010; 38(6):385-92. · 0.84 Impact Factor
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ABSTRACT: 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.
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.
Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies.
PLoS ONE 01/2010; 5(4):e9959. · 4.09 Impact Factor
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ABSTRACT: 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.
American Journal Of Pathology 11/2009; 175(6):2668-75. · 4.89 Impact Factor
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ABSTRACT: 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.
Journal of Cellular Physiology 06/2008; 215(2):497-505. · 3.87 Impact Factor
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ABSTRACT: Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short-ear (se) mice, which have BMP-5 mutations, a role for BMP-5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP-5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP-5 or its inhibition by anti-BMP-5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP-5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP-5 expression increasing as cells differentiated, in contrast to other BMPs. BMP-5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP-5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho-Smads. Anti-BMP-5 antibody inhibited the endogenous BMP-5, reducing cell proliferation and phospho-Smad nuclear accumulation. Together, the results demonstrate that BMP-5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP-5 loss-of-function mutations is discussed.
Journal of Cellular Physiology 02/2008; 214(1):56-64. · 3.87 Impact Factor
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Jian-Ming Li,
Xueqing Zhang,
Peter R Nelson, Paul R Odgren,
Janice D Nelson,
Calin Vasiliu,
Jane Park,
Marvin Morris,
Jane Lian,
Bruce S Cutler,
Peter E Newburger
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ABSTRACT: 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.
Journal of Cellular Biochemistry 06/2007; 101(2):399-410. · 2.87 Impact Factor
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Liesbeth Van Wesenbeeck, Paul R Odgren,
Fraser P Coxon,
Annalisa Frattini,
Pierre Moens,
Bram Perdu,
Carole A MacKay,
Els Van Hul,
Jean-Pierre Timmermans,
Filip Vanhoenacker,
Ruben Jacobs,
Barbara Peruzzi,
Anna Teti,
Miep H Helfrich,
Michael J Rogers,
Anna Villa,
Wim Van Hul
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ABSTRACT: 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.
Journal of Clinical Investigation 04/2007; 117(4):919-30. · 15.39 Impact Factor
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ABSTRACT: Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1(tl)/csf1(tl)) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.
Journal of Biological Chemistry 09/2006; 281(33):23598-605. · 4.77 Impact Factor
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ABSTRACT: Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor kappaB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1-treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.
Blood 04/2006; 107(6):2262-70. · 9.90 Impact Factor
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ABSTRACT: 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.
Connective Tissue Research 02/2006; 47(4):229-34. · 1.20 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Bone 11/2005; 37(4):530-44. · 4.02 Impact Factor
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Nacksung Kim,
Yuho Kadono,
Masamichi Takami,
Junwon Lee,
Seoung-Hoon Lee,
Fumihiko Okada,
Jung Ha Kim,
Takashi Kobayashi, Paul R Odgren,
Hiroyasu Nakano,
Wen-Chen Yeh,
Sun-Kyeong Lee,
Joseph A Lorenzo,
Yongwon Choi
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ABSTRACT: 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.
Journal of Experimental Medicine 10/2005; 202(5):589-95. · 13.85 Impact Factor
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ABSTRACT: 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.
Cytokine 08/2005; 31(2):94-102. · 3.02 Impact Factor
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Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 11/2004; 19(12):2093 - 2094. · 6.04 Impact Factor
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ABSTRACT: The incisors absent rat is an osteopetrotic animal model. Segregation analysis in 37 affected animals from an outcross enabled us to assign the disease causing gene to a 4.7-cM interval on rat chromosome 10q32.1. Further analysis of the genes mapped in this region will provide more insight into the underlying pathogenesis.
Many of the insights into the factors that regulate the differentiation and activation of osteoclasts are gained from different spontaneous and genetically induced osteopetrotic animal models. The osteopetrotic incisors absent (ia) rat exhibits a generalized skeletal sclerosis and a delay of tooth eruption. Although the ia rat has well been studied phenotypically, the genetic defect still remains unknown.
To map the ia locus, we outcrossed the inbred ia strain with the inbred strain Brown Norway. Intercrossing F1 animals produced the F2 generation. Thirty-one mutant F2 animals and six mutant F4 animals were available for segregation analysis.
Segregation analysis enabled us to assign the disease causing gene to rat chromosome 10q32.1. Homozygosity for the ia allele was obtained for two of the markers analyzed (D10Rat18 and D10Rat84). Key recombinations delineate a candidate region of 4.7 cM flanked by the markers D10Rat99 and D10Rat17.
We have delineated a 4.7-cM region on rat chromosome 10q32.1 in which the gene responsible for the osteopetrotic phenotype of the ia rat is located. Although the sequence of this chromosomal region is not complete, over 140 known or putative genes have already been assigned to this region. Among these, several candidate genes with a putative role in osteoclast functioning can be identified. However, at this point, it cannot be excluded that one of the genes with a currently unknown function is involved in the pathogenesis of the ia rat. Further analysis of the genes mapped in this region will provide us more insight into the pathogenesis of this osteopetrotic animal model.
Journal of Bone and Mineral Research 03/2004; 19(2):183-9. · 6.37 Impact Factor
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ABSTRACT: The proliferation and hypertrophy of growth plate chondrocytes set the pace and pattern for growth of endochondral bones. Complex signaling pathways regulating chondrocyte differentiation during development and growth have been discovered in recent years, but as yet little is known about how chondrocytes are able to orient themselves to align properly with respect to the direction of bone growth. Histomorphometric methods developed for analysis of growth plates rely to a significant extent on assessments of the relative heights of the zones of proliferating and hypertrophic chondrocytes. In a growing number of osteopetrotic mutations, however, it is becoming apparent that growth plates lack clearly demarcated zones of chondrocyte differentiation, and they show other notable histological abnormalities that cannot be measured with standard approaches. This is particularly true of mutations in which osteoclasts are altogether absent. We therefore developed a new approach that measures the salient features of this type of chondrodysplasia and have applied it to the toothless (tl) rat. The tl rat has a frameshift mutation in the Csf-1 gene that renders it null, resulting in severe osteopetrosis. An accompanying pathology is a severe, progressive growth plate chondrodysplasia. We measured cell orientation, cell area, and local columnar organization as functions of distance from the upper margin of the growth plate, in addition to growth plate thickness and cell density. All these parameters were markedly abnormal in the tl rats, thus implicating Csf-1 not only in its well-established role in regulating osteoclastic bone resorption, but also in endochondral ossification. This approach should prove useful in distinguishing among growth plate chondrodysplasias, most especially in the growing number of osteopetrotic mutations having growth plates that lack the normal zonal organization and in which the chondrocytes are mis-oriented. In turn, detailed assessments of chondrocyte misorientation may give insights into how they normally are able to arrange themselves with such precision.
Connective Tissue Research 02/2004; 45(1):1-10. · 1.20 Impact Factor
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ABSTRACT: Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti-CTGF neutralizing antibody caused a dose-dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.
Journal of Cellular Physiology 08/2003; 196(1):51-62. · 3.87 Impact Factor
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ABSTRACT: We report the skeletal manifestations of restoring RANKL (TNFSF11/TRANCE; see foot note on nomenclature) expression in null mice using a lymphocyte-specific promoter. RANKL was discovered independently by immunologists and bone researchers by virtue of its essential roles in lymph node organogenesis, normal cellular immunity, and osteoclastogenesis. "Rescue" of RANKL knockout mice by a T- and B-cell expressed transgene reversed many immunological manifestations of the knockout, while it had highly selective effects on the skeletal pathology. RANKL-null mice exhibit severe osteopetrosis, no tooth eruption, markedly reduced skeletal growth, and growth plate chondrodystrophy. The transgene induced tartrate-resistant acid phosphatase (TRAP) positive cells in long bones as early as 3 days postpartum, restored marrow spaces in long bones, produced lamellar bone in the diaphyses, and restored osteoclasts at many endosteal sites, but not in periosteum nor the jaws. It did not improve the chondrodystrophy, chondroosseous junction defects, or tooth eruption. The ends of limb and axial skeletal elements remained highly sclerotic while diaphyses became osteopenic, and growth retardation persisted. Together, these results demonstrate the importance of local delivery of RANKL for many skeletal processes.
Connective Tissue Research 02/2003; 44 Suppl 1:264-71. · 1.20 Impact Factor
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ABSTRACT: A "bone" is really a dynamic and highly interactive complex of many cell and tissue types. In particular, for the majority of skeletal elements to develop and grow, the process of endochondral ossification requires a constantly moving interface between cartilage, invading blood vessels, and bone. A great deal has been learned in recent years about the regulation of chondrocyte proliferation and differentiation by hormones, growth factors, and physiologic stimuli during skeletal development and growth. Likewise, the discovery that colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, a tumor necrosis factor superfamily member also called TRANCE, ODF, OPGL, and TNFSF11) are pivotal in communicating from osteoblasts to osteoclasts has led to deeper insights into bone growth, turnover, and maintenance. Little is known, however, about how these two quite different systems communicate to solve the problem of providing integrated, continuous mechanical support during the dynamic invasion of cartilage by bone that characterizes endochondral bone growth. Evidence has accumulated in recent years that provides insight into the communication between growing bone and cartilage in the form of a subset of osteopetrotic mutations, which share a lack of osteoclasts and an accompanying chondrodysplasia of the growth plate. These mutations thus implicate some of the same gene products in regulating chondrocyte differentiation and bone resorption. We also consider expression studies of some known growth plate regulators, such as parathyroid hormone-related protein (PTHrP) and Indian hedgehog (Ihh), in light of this and propose a model in which the osteoclastogenic factors act also on chondrocytes, but downstream of PTRrP and Ihh in regulating proliferation and differentiation, and after early morphogenic patterns are established.
Critical Reviews in Eukaryotic Gene Expression 02/2003; 13(2-4):181-93. · 3.08 Impact Factor
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ABSTRACT: The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a truncated, nonfunctional protein and an early stop codon, thus rendering the tl rat CSF-1(null). All mutants were homozygous for the mutation and all carriers were heterozygous. No CSF-1 transcripts were identified in rat mRNA that would avoid the mutation via alternative splicing. The biology and actions of CSF-1 have been elucidated by many studies that use another naturally occurring mutation, the op mouse, in which a single base insertion also disrupts the reading frame. The op mouse has milder osteoclastopenia and osteopetrosis than the tl rat and recovers spontaneously over the first few months of life. Thus, the tl rat provides a second model in which the functions of CSF-1 can be studied. Understanding the similarities and differences in the phenotypes of these two models will be important to advancing our knowledge of the many actions of CSF-1.
Proceedings of the National Academy of Sciences 11/2002; 99(22):14303-8. · 9.68 Impact Factor
