Polyphenols from persimmon fruit attenuate acetaldehyde-induced DNA double-strand breaks by scavenging acetaldehyde.

Acetaldehyde, a metabolic product of ethanol, induces DNA damage and genome instability. Accumulation of acetaldehyde due to alcohol consumption or aldehyde dehydrogenase (ALDH2) deficiency increases the risks of various types of cancers, including esophageal cancer. Although acetaldehyde chemically induces DNA adducts, the repair process of the lesions remains unclear. To investigate the mechanism of repair of acetaldehyde-induced DNA damage, we determined the repair pathway using siRNA knockdown and immunofluorescence assays of repair factors. Herein, we report that acetaldehyde induces DNA double-strand breaks (DSBs) in human U2OS cells and that both DSB repair pathways, non-homologous end-joining (NHEJ) and homology-directed repair (HDR), are required for the repair of acetaldehyde-induced DNA damage. Our findings suggest that acetaldehyde-induced DNA adducts are converted into DSBs and repaired via NHEJ or HDR in human cells. To reduce the risk of acetaldehyde-associated carcinogenesis, we investigated potential strategies of reducing acetaldehyde-induced DNA damage. We report that polyphenols extracted from persimmon fruits and epigallocatechin, a major component of persimmon polyphenols, attenuate acetaldehyde-induced DNA damage without affecting the repair kinetics. The data suggest that persimmon polyphenols suppress DSB formation by scavenging acetaldehyde. Persimmon polyphenols can potentially inhibit carcinogenesis following alcohol consumption.

FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway.

Microsatellites are short tandem repeat sequences that are highly prone to expansion/contraction due to their propensity to form non-B-form DNA structures, which hinder DNA polymerases and provoke template slippage. Although error correction by mismatch repair plays a key role in preventing microsatellite instability (MSI), which is a hallmark of Lynch syndrome, activities must also exist that unwind secondary structures to facilitate replication fidelity. Here, we report that Fancj helicase-deficient mice, while phenotypically resembling Fanconi anemia (FA), are also hypersensitive to replication inhibitors and predisposed to lymphoma. Whereas metabolism of G4-DNA structures is largely unaffected in Fancj(-/-) mice, high levels of spontaneous MSI occur, which is exacerbated by replication inhibition. In contrast, MSI is not observed in Fancd2(-/-) mice but is prevalent in human FA-J patients. Together, these data implicate FANCJ as a key factor required to counteract MSI, which is functionally distinct from its role in the FA pathway.

HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis.

Repair of interstrand crosslinks (ICLs) requires the coordinated action of the intra-S-phase checkpoint and the Fanconi anaemia pathway, which promote ICL incision, translesion synthesis and homologous recombination (reviewed in refs 1, 2). Previous studies have implicated the 3'-5' superfamily 2 helicase HELQ in ICL repair in Drosophila melanogaster (MUS301 (ref. 3)) and Caenorhabditis elegans (HELQ-1 (ref. 4)). Although in vitro analysis suggests that HELQ preferentially unwinds synthetic replication fork substrates with 3' single-stranded DNA overhangs and also disrupts protein-DNA interactions while translocating along DNA, little is known regarding its functions in mammalian organisms. Here we report that HELQ helicase-deficient mice exhibit subfertility, germ cell attrition, ICL sensitivity and tumour predisposition, with Helq heterozygous mice exhibiting a similar, albeit less severe, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralogue complex BCDX2 and functions in parallel to the Fanconi anaemia pathway to promote efficient homologous recombination at damaged replication forks. Thus, our results reveal a critical role for HELQ in replication-coupled DNA repair, germ cell maintenance and tumour suppression in mammals.

1-Alpha, 25-dihydroxy vitamin D3 inhibits osteoclastogenesis through IFN-beta-dependent NFATc1 suppression.

1-Alpha, 25-dihydroxy vitamin D(3) (1alpha,25(OH)(2)D(3)), an active form of vitamin D(3), plays a critical role in calcium and bone metabolism. Although 1alpha,25(OH)(2)D(3) has been used for osteoporosis therapy, the direct role of 1alpha,25(OH)(2)D(3) on human osteoclastogenesis has not been well characterized. Here we show that 1alpha,25(OH)(2)D(3) treatment significantly inhibited human osteoclast formation at the early stage of differentiation in a concentration-dependent manner. 1alpha,25(OH)(2)D(3) inhibited the expression of nuclear factor of activated T cells c1 (NFATc1, also referred as NFAT2), an essential transcription factor for osteoclast differentiation, and upregulated the expression of interferon-beta (IFN-beta), a strong inhibitor of osteoclastogenesis in osteoclast progenitors. Inhibitory effects of 1alpha,25(OH)(2)D(3) on osteoclastogenesis and NFATc1 expression were restored by treatment with an antibody against IFN-beta, suggesting that upregulation of IFN-beta by 1alpha,25(OH)(2)D(3) treatment results in inhibition of NFATc1 expression, in turn interfering with osteoclast formation. Thus, our study may provide a molecular basis for the treatment of human bone diseases by 1alpha,25(OH)(2)D(3) through regulation of the IFN-beta and NFATc1 axis.

Cell surface colony-stimulating factor 1 can be cleaved by TNF-alpha converting enzyme or endocytosed in a clathrin-dependent manner.

CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-alpha converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.

A novel promoter regulates calcitonin receptor gene expression in human osteoclasts.

The calcitonin receptor (CTR) is expressed in a wide variety of tissues and cell types. In bone, its expression is restricted to osteoclasts, the cells that mediate bone resorption. The human CTR (hCTR) gene has a complex structural organization that exhibits similarity to the porcine (pCTR) and mouse (mCTR) CTR genes. In these species, alternative splicing of a single gene generates multiple CTR isoforms that are distributed in both tissue-specific and species-specific patterns. However, the structural organization of the 5' putative regulatory region and transcriptional mechanisms responsible for tissue-specific expression of the different CTR isoforms are not fully defined. The present studies were undertaken to characterize the structural organization of the 5'-region of the hCTR and identify the regulatory regions involved in osteoclast-specific transcriptional activation. Analysis of mRNA prepared from human osteoclasts using reverse transcription-polymerase chain reaction (RT-PCR) and transient transfection of hCTR promoter-luciferase reporter constructs identified two regions in the 5'-flanking sequence of the hCTR gene that regulated CTR gene expression in osteoclasts. Both of these putative promoters were responsive to the osteoclast-inducing cytokine, receptor activator of NF-kappaB ligand (RANKL) and demonstrated trans-activation by the RANKL-induced transcription factor nuclear factor of activated T cells (NFATc1), consistent with a role in regulating CTR gene expression in osteoclasts.

The role played by cell-substrate interactions in the pathogenesis of osteoclast-mediated peri-implant osteolysis.

Prosthetic wear debris-induced peri-implant osteolysis is a major cause of aseptic loosening after total joint replacement. In this condition, wear particles released from the implant components induce a granulomatous inflammatory reaction at the interface between implant and adjacent bone, leading to progressive bone resorption and loss of fixation. The present study was undertaken to characterize definitively the phenotype of osteoclast-like cells associated with regions of peri-implant focal bone resorption and to compare the phenotypic features of these cells with those of mononucleated and multinucleated cells associated with polyethylene wear particles. Peri-implant tissues were obtained from patients undergoing hip revision surgery for aseptic loosening after total joint replacement. Cells were examined for the expression of several markers associated with the osteoclast phenotype using immunohistochemistry, histochemistry, and/or in situ hybridization. CD68 protein, a marker expressed by multiple macrophage lineage cell types, was detected in mononucleated and multinucleated cells associated with polyethylene particles and the bone surface. Cathepsin K and tartrate-resistant acid phosphatase were expressed highly in both mononucleated and multinucleated cells associated with the bone surface. Levels of expression were much lower in cells associated with polyethylene particles. High levels of beta3 integrin protein were detected in cells in contact with bone. Multinucleated cells associated with polyethylene particles exhibited faint positive staining. Calcitonin receptor mRNA expression was detected solely in multinucleated cells present in resorption lacunae on the bone surface and was absent in cells associated with polyethylene particles. Our findings provide further evidence that cells expressing the full repertoire of osteoclast phenotypic markers are involved in the pathogenesis of peri-implant osteolysis after total joint replacement. They also demonstrate that foreign body giant cells, although believed to be phenotypically and functionally distinct from osteoclasts, express many osteoclast-associated genes and gene products. However, the levels and patterns of expression of these genes in the two cell types differ. We speculate that, in addition to the role of cytokines and growth factors, the substrate with which these cells interact plays a critical role in their differential phenotypic and functional properties.