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.

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.

NHA-oc/NHA2: a mitochondrial cation-proton antiporter selectively expressed in osteoclasts.

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.

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor-beta1 (TGF-beta1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF-beta1-induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF-beta1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF-beta1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF-specific siRNA was used to prevent TGF-beta1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up-regulation. To examine the effects of TGF-beta1 on osteoblast cell growth, cultures were treated with TGF-beta1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF-beta1-induced cell-cycle arrest. Cultures transfected with CTGF siRNA prior to TGF-beta1 treatment showed an even greater reduction in cell number, suggesting that TGF-beta1-induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF-beta1-induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation.

CSF-1 regulation of osteoclastogenesis for tooth eruption.

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.

Growth hormone substitution increases gene expression of members of the IGF family in cortical bone from women with adult onset growth hormone deficiency--relationship with bone turn-over.

OBJECTIVE: To investigate the effects of growth hormone (GH) replacement therapy on bone matrix gene expression of insulin-like growth factors (IGFs) and markers of bone metabolism in women with adult-onset GH deficiency (GHD). DESIGN AND METHODS: Nineteen women, mean age 45 (range 24-56) years, were included in a double-blind, placebo-controlled parallel group study for 12 months. Biochemical markers were measured at baseline, 6 and 12 months. Bone biopsies were obtained and BMD was measured at baseline and after 12 months. RESULTS: Maximum responses were observed after 6 and 12 months, for bone resorptive and bone formative markers respectively. GH therapy enhanced gene expression in cortical bone of IGFs, GH-and calcitonin-receptor (CR) and osteoprotegerin (OPG), however with the most pronounced effects on CR and IGF-I. Changes in IGF-I gene expression during longitudinal follow-up were significantly correlated with changes in both circulating IGF-I (r = 0.82, p < 0.05), changes in markers of enhanced osteoclastic activity, measured both locally in bone (CR, r = 0.87, p < 0.01) and in serum (CTX-I, r = 0.86, p < 0.05), as well as serum bone ALP (r = 0.96, p < 0.01). CONCLUSIONS: This study indicates that both liver- and bone-derived IGF-I may be significant in mediating the effects of GH on bone metabolism in humans.

Evidence that the rat osteopetrotic mutation toothless (tl) is not in the TNFSF11 (TRANCE, RANKL, ODF, OPGL) gene.

The toothless (tl) osteopetrotic mutation in the rat affects an osteoblast-derived factor that is required for normal osteoclast differentiation. Although the genetic locus remains unknown, the phenotypic impact of the tl mutation on multiple systems has been well characterized. Some of its actions are similar to tumornecrosis factor superfamily member 11(TNFSF11; also called TRANCE, RANKL, ODF and OPGL) null mice. TNFSF11 is a recently described member of the tumor necrosis factor superfamily which, when expressed by activated T cells, enhances the survival of antigen-presenting dendritic cells, and when expressed by osteoblasts, promotes the differentiation and activation of osteoclasts. The skeletal similarities between tl rats and TNFSF11(-/-) mice include 1) profound osteoclastopenia (TNFSF11-null mice, 0% and tl rats 0-1% of normal); 2) persistent, non-resolving osteopetrosis that results from 3) a defect not in the osteoclast lineage itself, but in an osteoblast-derived, osteoclastogenic signal; and 4) a severe chondrodysplasia of the growth plates of long bones not seen in other osteopetrotic mutations. The latter includes thickening of the growth plate with age, disorganization of chondrocyte columns, and disturbances of chondrocyte maturation. These striking similarities prompted us to undertake studies to rule in or out a TNFSF11 mutation in the tl rat. We looked for expression of TNFSF11 mRNA in tl long bones and found it to be over-expressed and of the correct size. We also tested TNFSF11 protein function in the tl rat. This was shown to be normal by flow cytometry experiments in which activated, spleen-derived T-cells from tl rats exhibited normal receptor binding competence, as measured by a recombinant receptor assay. We also found that tl rats develop histologically normal mesenteric and peripheral lymph nodes, which are absent from TNFSF11-null mice. Next, we found that injections of recombinant TNFSF11, which restores bone resorption in null mice, had no therapeutic effect in tl rats. Finally, gene mapping studies using co-segregation of polymorphic markers excluded the chromosomal region containing the TNFSF11 gene as harboring the mutation responsible for the tl phenotype. We conclude that, despite substantial phenotypic similarities to TNFSF11(-/-) mice, the tl rat mutation is not in the TNFSF11 locus, and that its identification must await the results of further studies.

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene.

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

Endochondral bone formation in toothless (osteopetrotic) rats: failures of chondrocyte patterning and type X collagen expression.

The pacemaker of endochondral bone growth is cell division and hypertrophy of chondrocytes. The developmental stages of chondrocytes, characterized by the expression of collagen types II and X, are arranged in arrays across the growth zone. Mutations in collagen II and X genes as well as the absence of their gene products lead to different, altered patterns of chondrocyte stages which remain aligned across the growth plate (GP). Here we analyze GP of rats bearing the mutation toothless (tl) which, apart from bone defects, develop a progressive, severe chondrodystrophy during postnatal weeks 3 to 6. Mutant GP exhibited disorganized, non-aligned chondrocytes and mineralized metaphyseal bone but without cartilage mineralization or cartilaginous extensions into the metaphysis. Expression of mRNA coding for collagen types II (Col II) and X (Col X) was examined in the tibial GP by in situ hybridization. Mutant rats at 2 weeks exhibited Col II RNA expression and some hypertrophied chondrocytes (HC) but no Col X RNA was detected. By 3rd week, HC had largely disappeared from the central part of the mutant GP and Col II RNA expression was present but weak and in 2 separate bands. Peripherally the GP contained HC but without Col X RNA expression. This abnormal pattern was exacerbated by the fourth week. Bone mineralized but cartilage in the GP did not. These data suggest that the tl mutation involves a regulatory function for chondrocyte maturation, including Col X RNA synthesis and mineralization, and that the GP abnormalities are related to the Col X deficiency. The differences in patterning in the tl rat GP compared to direct Col X mutations may be explained by compensatory effects.

Cloning the full-length cDNA for rat connective tissue growth factor: implications for skeletal development.

The mammalian osteopetroses represent a pathogenetically diverse group of skeletal disorders characterized by excess bone mass resulting from reduced osteoclastic bone resorption. Abnormalities involving osteoblast function and skeletal development have also been reported in many forms of the disease. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between op mutants and their normal littermates. RNA isolated from calvaria and long bones was used as a template for mRNA-differential display. Sequence information for one of the many cDNA that were selectively expressed in either normal or mutant bone suggested that it is the rat homologue of connective tissue growth factor (CTGF) previously cloned in the human, mouse, and other species. A consensus sequence was assembled from overlapping 5'-RACE clones and used to confirm the rat CTGF cDNA protein coding region. Northern blot analysis confirmed that this message was highly (8- to 10-fold) over-expressed in op versus normal bone; it was also upregulated in op kidney but none of the other tissues (brain, liver, spleen, thymus) examined. In primary rat osteoblast cultures, the CTGF message exhibits a temporal pattern of expression dependent on their state of differentiation. Furthermore, CTGF expression is regulated by prostaglandin E(2), a factor known to modulate osteoblast differentiation. Since members of the CTGF family regulate the expression of specific genes, such as collagen and fibronectin, we propose that CTGF may play a previously unreported role in normal skeletal modeling/remodeling. Its dramatic over-expression in the op mutant skeleton may be secondary to the uncoupling of bone resorption and bone formation resulting in dysregulation of osteoblast gene expression and function.

Sutures, growth plates and the craniofacial base--experimental studies in the toothless (tl-osteopetrotic) rat.

The craniofacial skeleton develops from a base in which coordinated growth at sutures and growth centres assures the development of normal form. In this report we describe features of retarded postnatal craniofacial development in the osteopetrotic mutation, toothless (tl), in the rat in which bone growth in both the nasal area and the cranial base is reduced, suggesting that the mutation affects bone formation in sutures and growth plates. We began a systematic search for potential mechanisms by analysing the expression in time and intensity of RNA coding for collagens type I (Col I) and type III (Col III) analysed by in situ hybridisation of cells in the premaxillary-maxillary suture (PMMS). In the centre of the PMMS of tl rats, cells expressing Col I and Col III appeared later than in normal littermates and exhibited lower signal. During osteoblast recruitment from the suture centre into the bone domains, Col III RNA expression is switched off. Osteoblasts expressing Col I in abundance, but no Col III, appeared in the flanking bone regions of tl rats later than in normal littermates. It is proposed that the tl mutation restricts the number of available osteoblast progenitor cells, and that the shortage of these cells affects bone growth in the PMMS and in the cranial base. Additional analyses are needed to test this hypothesis and to understand the developmental dynamics in the cranial base.

The toothless osteopetrotic rat has a normal vitamin D-binding protein-macrophage activating factor (DBP-MAF) cascade and chondrodysplasia resistant to treatments with colony stimulating factor-1 (CSF-1) and/or DBP-MAF.

The osteopetrotic rat mutation toothless (tl) is characterized by little or no bone resorption, few osteoclasts and macrophages, and chondrodysplasia at the growth plates. Short-term treatment of tl rats with colony-stimulating factor-1 (CSF-1) has been shown to increase the number of osteoclasts and macrophages, producing dramatic resolution of skeletal sclerosis at some, but not all, sites. Defects in production of vitamin D-binding protein-macrophage activating factor (DBP-MAF) have been identified in two other independent osteopetrotic mutations of the rat (op and ia), and two in the mouse (op and mi), in which macrophages and osteoclasts can be activated by the administration of exogenous DBP-MAF. The present studies were undertaken to examine the histology and residual growth defects in tl rats following longer CSF-1 treatments, to investigate the possibility that exogenous DBP-MAF might act synergistically with CSF-1 to improve the tl phenotype, and to assess the integrity of the endogenous DBP-MAF pathway in this mutation. CSF-1 treatment-with or without DBP-MAF-induced resorption of metaphyseal bone to the growth plate on the marrow side, improved slightly but did not normalize long bone growth, and caused no improvement in the abnormal histology of the growth plate. Injections of lysophosphatidylcholine (lyso-Pc) to prime macrophage activation via the DBP-MAF pathway raised superoxide production to similar levels in peritoneal macrophages from both normal and mutant animals, indicating no defect in the DBP-MAF pathway in tl rats. Interestingly, pretreatments with CSF-1 alone also increased superoxide production, although the mechanism for this remains unknown. In summary, we find that, unlike other osteopetrotic mutations investigated to date, the DBP-MAF pathway does not appear to be defective in the tl rat; that additional DBP-MAF does not augment the beneficial skeletal effects seen with CSF-1 alone; and that the growth plate chondrodystrophy seen in this mutation is unaffected by either molecule. Thus, the tl mutation intercepts the function of a gene required for both normal endochondral ossification and bone resorption, thereby uncoupling the coordination of skeletal metabolism required for normal long bone growth.

Facial development and type III collagen RNA expression: concurrent repression in the osteopetrotic (Toothless,tl) rat and rescue after treatment with colony-stimulating factor-1.

The toothless (osteopetrotic) mutation in the rat is characterized by retarded development of the anterior facial skeleton. Growth of the anterior face in rats occurs at the premaxillary-maxillary suture (PMMS). To identify potential mechanisms for stunted facial growth in this mutation we compared the temporospatial expression of collagen I (Col I) and collagen III (Col III) RNA around this suture in toothless (tl) rats and normal littermates by in situ hybridization of specific riboprobes in sagittal sections of the head. In normal rats, the suture is S shaped at birth and becomes highly convoluted by 10 days with cells in the center (fibroblasts and osteoblast progenitors) expressing Col III RNA and those at the periphery (osteoblasts) expressing no Col III RNA but high amounts of Col I RNA throughout the growth phase (the first 2 postnatal weeks). In the mutant PMMS, cells were reduced in number, less differentiated, and fewer osteoblasts were encountered. Expression of Col I RNA was at normal levels, but centrosutural cells expressed Col III RNA only after day 6 and then only weakly. A highly convoluted sutural shape was never achieved in mutants during the first 2 postnatal weeks. Treatment of tl rats with the cytokine CSF-1 improved facial growth and restored cellular diversity and Col III RNA expression in the PMMS to normal levels. Taken together, these data suggest that normal facial growth in rats is related to expression of Col III RNAby osteoblast precursors in the PMMS, that these cells are deficient in the tl mutation and are rescued following treatment with CSF-1.

Cellular and molecular strategies for studying the regulation of bone resorption using the toothless (osteopetrotic) mutation in the rat.

The division of labor among cells of the skeleton is distinct and diverse and the regulation of these cells is interdependent. Osteoclasts are the cellular source of bone resorption and signals for their development and activation come, at least in part, from bone and other cells in the local environment. Studies of isolated cells have identified some factors in the developmental cascade of osteoclasts but there is little understanding of the sequence and local concentrations, not to mention other factors, needed for both the development of competent osteoclasts and for coordinated bone resorption. We review the skeletal biology of one osteopetrotic mutation in the rat, toothless, in which bone resorption is severely reduced because of a failure in the development and function of osteoclasts. Furthermore, we review the advantages and limitations of a relatively new method, differential display of mRNA (DD), that identifies differences in gene expression in two or more populations of cells. We present a strategy and preliminary data for the application of DD to this mutation. We propose that application of this method to these and other skeletal diseases, with the appropriate controls and confirmations, will provide data about pathogenetic pathways and has a high probability for identifying new regulators of skeletal development and turnover.

Topoisomerase II expression in osseous tissue.

The molecular mechanisms that mediate the transition from an osteoprogenitor cell to a differentiated osteoblast are unknown. We propose that topoisomerase II (topo II) enzymes, nuclear proteins that mediate DNA topology, contribute to coordinating the loss of osteoprogenitor proliferative capacity with the onset of differentiation. The isoforms topo II-alpha and -beta, are differentially expressed in nonosseous tissues. Topo II-alpha expression is cell cycle-dependent and upregulated during mitogenesis. Topo II-beta is expressed throughout the cell cycle and upregulated when cells have plateaued in growth. To determine whether topo II-alpha and -beta are expressed in normal bone, we analyzed rat lumbar vertebrae using immunohistochemical staining. In the tissue sections, topo II-alpha was expressed in the marrow cavity of the primary spongiosa. Mature osteoblasts along the trabecular surfaces did not express topo II-alpha, but were immunopositive for topo II-beta, as were cells of the marrow cavity. Confocal laser scanning microscopy was used to determine the nuclear distribution of topo II in rat osteoblasts isolated from the metaphyseal distal femur and the rat osteosarcoma cells, ROS 17/2.8. Topo II-alpha exhibited a punctate nuclear distribution in the bone cells. Topo II-beta was dispersed throughout the interior of the nucleus but concentrated at the nuclear envelope. Serum starvation of the cells attenuated topo II-alpha expression but did not modulate expression of the beta-isoform. These results indicate that the loss of osteogenic proliferation correlates with the downregulation of topo II-alpha expression.

Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains.

We have identified and characterized a human protein of the mitochondria which we call mitofilin. Using monoclonal and polyclonal antibodies, we have isolated cDNA clones and characterized mitofilin biochemically. It appears as a 90 and 91 kDa doublet in western blots and is translated from a single 2.7 kb mRNA. Antibodies raised against cellular and bacterially-expressed protein given identical cytoplasmic immunofluorescence and immunoblot results. Mitofilin co-localizes with mitochondria in immunofluorescence experiments and co-purifies with mitochondria. Double label studies show co-localization only with mitochondria and not with Golgi or endoplasmic reticulum. Co-localization with mitochondria is retained when actin or tubulin are de-polymerized, and mitofilin is expressed in all human cell types tested. The cDNA encodes a polypeptide with a central alpha-helical region with predicted coiled coil domains flanked by globular amino and carboxy termini. Unlike coiled coil motor proteins, mitofilin is resistant to detergent extraction. The presence of mitochondrial targeting and stop-transfer sequences, along with the accessibility of mitofilin to limited proteolysis suggests that it resides predominantly in the intermembrane space, consistent with immuno-electron micrographs which show mitofilin mainly at the mitochondrial periphery. The cDNA sequence of mitofilin is identical to that recently reported by Icho et al. (1994; Gene 144, 301-306) for a mRNA preferentially expressed in heart muscle (HMP), consistent with the high levels of mitochondria in cardiac myocytes.

Thrombocytopenia in the toothless (osteopetrotic) rat and its rescue by treatment with colony-stimulating factor-1.

Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover.

Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex.

We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen lambda gt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from 32P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed.

Phylogenetic occurrence of coiled coil proteins: implications for tissue structure in metazoa via a coiled coil tissue matrix.

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.

Sp1 trans-activation of cell cycle regulated promoters is selectively repressed by Sp3.

The transcription factor Sp1 plays a key role in the activation of many cellular and viral gene promoters, including those that are regulated during the cell cycle. However, recent evidence indicates that Sp1 belongs to a larger family of factors which bind G/C box elements in order to either activate or repress transcription. Sp3, a member of this family, functions to repress transcriptional activation in two viral promoters, most likely by competing with Sp1 for GC box/Sp binding sites. However, the physiological role of Sp3 in the repression of endogenous cellular promoters has not been experimentally addressed. In the present study, we analyze the activity and binding of Sp3 on several eukaryotic promoters that contain G/C boxes and are known to be regulated during cellular proliferation and the cell cycle. Using antibodies specific for Sp1 and Sp3, we observe that both of these factors localize to the cell nucleus and have a similar, dispersed subnuclear distribution. Further, using gel mobility shift assays, we show that both Sp1 and Sp3 interact specifically with the histone H4 promoter. Transient cotransfections of Drosophila cells with Sp1 and Sp3 expression vectors and with the histone H4, thymidine kinase (TK), or dihydrofolate reductase (DHFR) promoters show that only the DHFR promoter, containing multiple functional GC boxes, displays Sp3 repression of Sp1 activation. In contrast, the single G/C boxes within the histone H4 or TK promoters, which confer transcriptional activation via Sp1 binding, are not responsive to repression by Sp3. Therefore, we demonstrate that the endogenous cellular DHFR promoter is selectively responsive to Sp3 repression. The data suggest that Sp3 may contribute to the control of proliferation- and/or cell-regulated promoters depending upon the context and/or number of functional Sp1 binding sites.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression.