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.     

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43^hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.     

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 and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.     

Transformations of osteoclast phenotype in ia rats cured of congenital osteopetrosis

The reduced bone resorption characteristic of osteopetrosis is accompanied in the incisors-absent (ia) rat mutation by a significant increase in osteoclasts of inactive (mutant) phenotype. Restoration of bone resorption in ia rats by transfer of spleen cells from normal littermates is preceded by a transformation of osteoclasts from mutant to normal phenotype. In this investigation the proportions of osteoclasts of normal phenotype have been determined by light microscopy in untreated ia and normal rats and in ia rats treated with various cell populations from normal rats. Significant increases in numbers of osteoclasts of normal phenotype were seen in the mutant skeleton soon after cell treatments that eventually restored bone resorption and cured the disease. No changes in osteoclast phenotype were seen after cell transfers that did not cure the disease. These data establish transformation of osteoclast phenotype as an early event in the recovery from osteopetrosis and suggest that determination of osteoclast phenotype is a reliable predictor of the success of normal cell populations to restore bone resorption in this mutation.     

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology.     

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.     

Gene expression during skeletal development in three osteopetrotic rat mutations. Evidence for osteoblast abnormalities

Osteopetrosis is a group of metabolic bone diseases characterized by reductions in osteoclast development and/or function. These aspects of osteoclast biology are known to be influenced by osteoblasts and their products. To ascertain whether osteoblast dysfunction contributes to aberrations in the structural and functional properties of osteoclasts in osteopetrosis, we systematically examined gene expression as reflected by mRNA levels for a series of cell growth- and tissue-related genes associated with the osteoblast phenotype during skeletal development in normal and mutant rats of three different osteopetrotic stocks. We show that the methods used permit the reproducible isolation of undegraded total cellular RNA from bone and that mRNA levels can be reliably quantitated in these preparations. Each osteopetrotic mutation exhibits a distinct aberrant pattern of osteoblast gene expression that may be correlated with and explain some abnormalities in extracellular matrix composition, mineralization, osteoclast development, and effects of elevated serum levels of 1 alpha,25-dihydroxyvitamin D3, depending upon the mutation. Normal rats show minor variations in gene expression that reflect the genetic background (stock). This, the first comprehensive molecular analysis of osteoblast gene expression in osteopetrosis, suggests that some osteopetroses, particularly in the toothless rat, are associated with and potentially related to mechanisms associated with aberrations in osteoblast function. More generally, the present studies demonstrate alterations in gene expression as reflected by mRNA levels that are associated with functional properties of the osteoblast, particularly those contributing to the recruitment and/or differentiation of osteoclasts, thereby influencing skeletal modeling.     

Cloning and mapping of a testis-specific gene with sequence similarity to a sperm-coating glycoprotein gene

A testis-specific gene Tpx-1, located between Pgk-2 and Mep-1 on mouse chromosome 17, was isolated from a cosmid clone, and its cDNA sequences were determined. The predicted coding sequence of Tpx-1 isolated from BALB/c mice showed 64.2% nucleotide and 55.1% amino acid sequence similarity with that of a rat sperm-coating glycoprotein gene, the protein product of which is secreted by the epididymis. To examine the evolutionary relationship between Tpx-1 and a sperm-coating glycoprotein gene, the cDNA sequence of TPX1, the human counterpart of Tpx-1, was determined. The comparison of the predicted coding sequences of Tpx-1 and TPX1 showed 77.8% nucleotide and 70% amino acid sequence similarity. Since Tpx-1 (from mouse) is more similar to TPX1 (from man) than it is to a rat sperm-coating glycoprotein gene, we conclude that Tpx-1 (TPX1) and a sperm-coating glycoprotein gene are closely related, but distinct, genes belonging to the same gene family. The predicted Tpx-1 protein of a t mutant mouse CRO437 differs from that of BALB/c mice by one amino acid insertion in the putative signal peptide. TPX1 was mapped to 6p21-qter by Southern blot analysis of interspecies somatic hybrid cell lines.     

Conditional deletion of the colony stimulating factor-1 receptor (c-fms proto-oncogene) in mice

Colony stimulating factor-1 (CSF-1) is the primary regulator of the mononuclear phagocytic lineage acting through its transmembrane tyrosine kinase receptor, CSF-1R, that is the product of the c-fms proto-oncogene. Null mutations in either the ligand or the receptor genes result in a severe osteopetrosis as well as a number of other phenotypes, including reproductive defects and perturbations in organ development. The CSF-1R is also expressed in oocytes, myoblast progenitors, decidual, and trophoblastic cells. To distinguish cell type specific phenotypes, we have created a conditional allele of the Csf1r by placing LoxP sites around Exon 5 of the Csf1r gene in mice. Excision of this floxed sequence results in a null allele that in the homozygous state gives a phenotype indistinguishable of the complete Csf1r null mutant mouse. This conditional allele will prove extremely valuable to study the spatial and temporal roles of CSF-1R.     

Carbonic anhydrase II deficiency: single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients.

To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. We report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical properties in long bones of rat osteopetrotic mutations.

Osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. Experimental studies with rat osteopetrotic mutations have shown increased bone density and decreased bone strength. However, it is not known if this reduction in bone strength is only due to changes in structure and geometry or if the tissue properties of bone material itself are changed as well. We have evaluated bone tissue properties with nanoindentation in three osteopetrotic mutations in the rat (incisors-absent ia/ia, osteopetrosis op/op and toothless tl/tl) to test the hypothesis that reduced bone resorption in these mutations results in reduced tissue properties of bone material. No significant differences in elastic modulus or hardness were found between osteopetrotic mutants and their normal littermates (NLMs) in any of the three stocks. This indicates that the tissue properties of bone material are not changed significantly in osteopetrosis, even if the mechanical strength is decreased at the macroscopic level.     

Skeletal biology in the toothless (osteopetrotic) rat

The toothless (tl) rat is a nonlethal osteopetrotic mutation characterized by the presence of few osteoclasts and the failure to be cured by bone-marrow transplantation. We examined the skeletal biology of tl rats and normal littermates up to 6 weeks after birth. Osteoclasts in tl rats were small, reduced 25-fold in number, and had greatly reduced concentrations of acid hydrolases. Bone shape internally and externally reflected reduced bone resorption, and tl rats were hypophosphatemic and mildly hypocalcemic at 2 weeks. These data indicate that the basic defect in tl rats is one of differentiation of osteoclasts and, coupled with the observation that normal bone-marrow cells cannot develop into osteoclasts in the tl skeleton, suggest that the defect lies in the skeletal micro-environment.     

Osteopetrosis in the toothless (t1) rat: presence of osteoclasts but failure to respond to parathyroid extract or to be cured by infusion of spleen or bone marrow cells from normal littermates.

Osteoclast have been observe for the first time in toothless (t1) rats, a mutation with inherits osteopetrosis as an autosomal recessive. The ability of t1 rats to raise the serum calcium concentration after injection of parathyroid extract was severely limited when compared with normal littermates. In addition, osteopetrosis in t1 rats is not cured by radiation and infusion of normal spleen or bone marrow cells from normal littermates, a method know to cure osteopetrosis in mutants of this and other species. This indirect evidence for a reduction in bone resorption as a cause of osteopetrosis in this mutation and the failure of transplanted cells to cure the disease are discussed in relation to the development and function of osteoclasts.     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

Cloning and expression of the rat interleukin-3 gene.

Genomic clones carrying the rat interleukin-3 (IL-3) gene have been isolated and the nucleotide sequence of the gene determined. Alignment of this sequence with that of the mouse IL-3 gene has allowed the structure of the rat IL-3 gene to be deduced. The intron-exon boundaries are conserved and extensive nucleotide homology (approx 90%) is present in the 5' flanking region and the portion of the gene coding for the signal peptide. Several proposed regulatory sequences are conserved and an analogous element to the tandem repeat in intron 2 of the mouse gene is also present. The predicted amino acid sequence for mature rat IL-3 shows surprisingly low homology (54%) with its murine counterpart, although all four cysteine residues are conserved. The rat IL-3 gene was expressed in monkey COS-1 cells and colony assays established that rat IL-3 is a multi-lineage haemopoietic growth regulator. There was little cross-reactivity of the respective IL-3 species on mouse and rat bone marrow cells suggesting that rat IL-3, in concert with its receptor, has evolved significantly away from the mouse IL-3/receptor system.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

A germ line mutation within the coding sequence for the putative 5-phosphoribosyl-1-pyrophosphate binding site of hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a Lesch-Nyhan patient: missense mutations within a functionally important region probably cause disease

Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.