Tumor necrosis factor-alpha (TNF) stimulates RANKL-induced osteoclastogenesis via coupling of TNF type 1 receptor and RANK signaling pathways.

Tumor necrosis factor-alpha (TNF) and the ligand for receptor activator of NF-kappaB (RANKL) are abundant in sites of inflammatory bone erosion. Because these cytokines are potent osteoclastogenic factors and because their signaling pathways are considerably overlapping, we postulated that under pro-inflammatory conditions RANKL and TNF might synergistically orchestrate enhanced osteoclastogenesis via cooperative mechanisms. We found TNF, via TNF type 1 receptor (TNFr1), prompts robust osteoclastogenesis by osteoclast precursors pretreated with RANKL, and deletion of TNFr1 abrogates this response. Enhanced osteoclastogenesis is associated with high expression of otherwise TNF and RANKL-induced mediators, including c-Src, TRAF2, TRAF6, and MEKK-1, levels of which were notably reduced in TNFr1 knockouts. Recruitment of TRAFs and MEKK1 leads to activation of downstream pathways, primarily I kappa B/NF-kappa B, ERKs, and cJun/AP-1. Consistent with impaired osteoclastogenesis and reduced expression of TRAFs and MEKK1, we found that phosphorylation and activation of I kappa B, NF-kappa B, ERKs, and cJun/AP-1 are severely reduced in RANKL-treated TNFr1-null osteoclast precursors compared with wild type counterparts. Finally, we found that TNF and RANKL synergistically up-regulate RANK expression in wild type precursors, whereas basal and stimulated levels of RANK are significantly lower in TNFr1 knockout cells. Our data suggest that exuberant TNF-induced osteoclastogensis is the result of coupling between RANK and TNFr1 and is dependent upon signals transmitted by the latter receptor.

Mice deficient in Abl are osteoporotic and have defects in osteoblast maturation.

The c-Abl protein is a non-receptor tyrosine kinase involved in many aspects of mammalian development. c-Abl kinase is widely expressed, but high levels are found in hyaline cartilage in the adult, bone tissue in newborn mice, and osteoblasts and associated neovasculature at sites of endochondrial ossification in the fetus. Mice homozygous for mutations in the gene encoding c-Abl (AIM) display increased perinatal mortality, reduced fertility, foreshortened crania and defects in the maturation of B cells in bone marrow. Here we demonstrate that Abl-/- mice are also osteoporotic. The long bones of mutant mice contain thinner cortical bone and reduced trabecular bone volume. The osteoporotic phenotype is not due to accelerated bone turnover--both the number and activity of osteoclasts are similar to those of control littermates--but rather to dysfunctional osteoblasts. In addition, the rate of mineral apposition in the mutant animals is reduced. Osteoblasts from both stromal and calvarial explants showed delayed maturation in vitro as measured by expression of alkaline phosphatase (ALP), induction of mRNA encoding osteocalcin and mineral deposition.

Substrate recognition by osteoclast precursors induces C-src/microtubule association.

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.

Osteoclasts, macrophages, and the molecular mechanisms of bone resorption.

The osteoclast is a physiological polykaryon and the major if not exclusive resorptive cell of bone. It participates in bone remodeling, repair, and growth and mobilization of mineral to meet homeostatic demands. Most importantly, osteoporosis, a disease endemic in Western society and Asia, is always a reflection of enhanced osteoclastic activity relative to bone formation by osteoblasts. In fact, all forms of anti-osteoporosis therapy proven successful involve inhibition of osteoclastic bone resorption. Bone resorption is regulated either by altering recruitment of osteoclast precursors into fully differentiated resorptive polykaryons or modulating the rate at which mature osteoclasts degrade bone. With this in mind, our laboratory has focused on the molecular mechanisms of osteoclast differentiation and the means by which the cell degrades bone matrix.

Osteopetrosis in mice lacking haematopoietic transcription factor PU.1.

Osteoclasts are multinucleated cells and the principal resorptive cells of bone. Although osteoclasts are of myeloid origin, the role of haematopoietic transcription factors in osteoclastogenesis has not been explored. Here we show that messenger RNA for the myeloid- and B-cell-specific transcription factor PU.1 progressively increases as marrow macrophages assume the osteoclast phenotype in vitro. The association between PU.1 and osteoclast differentiation was confirmed by demonstrating that PU.1 expression increased with the induction of osteoclastogenesis by either 1,25-dihydroxyvitamin D3 or dexamethasone. Consistent with the participation of PU.1 in osteoclastogenesis, we found that the development of both osteoclasts and macrophages is arrested in PU.1-deficient mice. Reflecting the absence of osteoclasts, PU.1-/- mice exhibit the classic hallmarks of osteopetrosis, a family of sclerotic bone diseases. These animals were rescued by marrow transplantation, with complete restoration of osteoclast and macrophage differentiation, verifying that the PU.1 lesion is intrinsic to haematopoietic cells. The absence of both osteoclasts and macrophages in PU.1-mutant animals suggests that the transcription factor regulates the initial stages of myeloid differentiation, and that its absence represents the earliest developmental osteopetrotic mutant yet described.

Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system.

Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.

The role of coiled-coil alpha-helices and disulfide bonds in the assembly and stabilization of cartilage matrix protein subunits. A mutational analysis.

Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.

rseB, a chromosomal locus that affects the stability of a temperature-specific surface protein mRNA in Tetrahymena thermophila.

In Tetrahymena thermophila, the expression of a temperature-specific surface protein known as SerH3 is primarily controlled by a temperature-dependent change in the stability of the mRNA that encodes this protein. At 30 degrees C the SerH3 mRNA displays a half-life of 60 minutes while at 40 degrees C the half-life decreases to only 3 minutes. We used a Tetrahymena mutant cell line (rseB) defective in expression of SerH3 at 30 degrees C to explore the mechanisms involved in temperature-dependent mRNA stability. The results of in vitro nuclear run-off assays and Northern and slot blot analysis of cytoplasmic and nuclear RNAs show that the rseB locus encodes a temperature-sensitive product that has no effect on SerH3 gene transcription or the steady-state levels of SerH3 nuclear RNA. However, the product of this locus does have a dramatic effect on cytoplasmic levels of the SerH3 mRNA at 30 degrees C, indicating that SerH3 gene expression is affected post-transcriptionally within the cytoplasm. To explore the possibility that the rseB locus controls SerH3 mRNA stability we developed an in vitro mRNA decay assay. This assay successfully duplicates the differential decay of the SerH3 mRNA observed in wild-type cells grown at different temperatures. The apparent half-life of the SerH3 mRNA in cytoplasmic extracts derived from cells grown at 30 degrees C is approximately 45 minutes while in cytoplasmic extracts derived from cells grown at 40 degrees C it is only 6 minutes. When similar experiments are performed using extracts prepared from the Tetrahymena rseB cell line, we find that the SerH3 mRNA is only stable in extract prepared from cells grown under conditions in which the mRNA accumulates to detectable levels in the cytoplasm. These results indicate that the product of the rseB locus is a trans-acting cytoplasmic factor that exerts its effect on SerH3 gene expression by regulating SerH3 mRNA stability.

Molecular characterization of SerH3, a Tetrahymena thermophila gene encoding a temperature-regulated surface antigen.

The DNA sequences of a cDNA clone and the macronuclear genomic fragment corresponding to the functional copy of the SerH3 surface antigen gene of Tetrahymena thermophila were determined. Primer extension and nuclease protection assays show that the SerH3 transcription unit is 1,425 nucleotides long and contains no introns. The predicted polypeptide encoded by the SerH3 gene has a molecular mass of 44,415 daltons; one-third of its 439 residues are either cysteine, serine, or threonine. The central half of the polypeptide consists of three homologous domains in tandem array; within these domains, the cysteine, proline, and tryptophan residues occur in highly regular patterns.

DNA rearrangements associated with the H3 surface antigen gene of Tetrahymena thermophila that occur during macronuclear development.

The surfaces of Tetrahymena thermophila cells grown between 20 and 35 degrees C are covered by one or more variants of H antigens. A cDNA clone, pC6, has previously been identified that hybridizes to a unique polyA+ RNA that appears to code for the SerH3 variant of the H antigens. pC6 and a subclone of it, pGpC6.295, were used to analyze the genomic organization of the corresponding gene(s) in both the macronucleus and the micronucleus. It was determined that pC6 hybridizes to a small family of sequences in the macronucleus, only one of which also hybridizes to pGpC6.295. The latter is a strong candidate for the gene encoding the SerH3 antigen. Sequences homologous to pC6 - but not to pGpC6.295 - are present in strains carrying the other SerH alleles. Shifts in antigen switching during vegetative growth do not result in any detectable DNA rearrangements in the vicinity of the pC6-hybridizing sequence family. Analysis of micronuclear DNA from a homozygous SerH3 strain revealed that it also contains a family of sequences that are homologous to pC6; but, in contrast to the macronuclear DNA, two members of this micronuclear sequence family hybridize to pGpC6.295. Comparison of micro- and macronuclear DNA indicate that some members of the pC6-positive sequence family rearrange during macronuclear development. These rearrangements fall into two classes: those which occur reproducibly, and those which show variability. The gene homologous to pGp6.295 falls into the former category.

Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes.

The ribosomal RNA genes (rDNA) of Tetrahymena thermophila macronucleus exist as free linear 21-kilobase molecules that contain replication origins and telomeres. A mutation in this gene confers resistance to the antibiotic paromomycin. We have isolated rDNA from such a mutant (strain p2f), microinjected it into the macronucleus of a sensitive strain, and obtained drug-resistant cells at a frequency of 1-3%. The transformed cells have a distinct and stable phenotype. The rDNA of the transformants contains the expected sequences of the mutant rDNA as determined by oligonucleotide hybridization. rDNA from a different inbred line (C3-368), which contains heteromorphic restriction sites, has also been used for injection, and the results confirm the fact that the injected rDNA is indeed present in the transformants. Injection of rDNA from the C3 strains also increases the transformation frequency 5- to 10-fold and leads to the total replacement of the resident rDNA of the B-inbred strains. This is presumably due to the replication dominance of rDNA from the C3 strains over that of the B strains. Using this method, we have also been able to transform developing cells, at similar frequencies, by microinjecting into the macronuclear anlagen.