Disequilibrium between bone-forming osteoblasts and bone-resorbing osteoclasts is central to many bone diseases. Here, we show that dysregulated expression of translationally controlled isoforms of CCAAT/enhancerbinding protein b (C/EBPb) differentially affect bone mass. Alternative translation initiation that is controlled by the mammalian target of rapamycin (mTOR) pathway generates long transactivating (LAP*, LAP) and a short repressive (LIP) isoforms from a single C/EBPb transcript. Rapamycin, an inhibitor of mTOR signalling increases the ratio of LAP over LIP and inhibits osteoclastogenesis in wild type (WT) but not in C/EBPb null (c/ebpb À/À ) or in LIP knock-in (L/L) osteoclast precursors. C/EBPb mutant mouse strains exhibit increased bone resorption and attenuated expression of MafB, a negative regulator of osteoclastogenesis. Ectopic expression of LAP and LIP in monocytes differentially affect the MafB promoter activity, MafB gene expression and dramatically affect osteoclastogenesis. These data show that mTOR regulates osteoclast formation by modulating the C/EBPb isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression.

Conserved upstream open reading frames (uORFs) are found within many eukaryotic transcripts and are known to regulate protein translation. Evidence from genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of human diseases. A genetic mouse model has recently provided proof-of-principle support for the physiological relevance of uORF-mediated translational control in mammals. The targeted disruption of the uORF initiation codon within the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) gene resulted in deregulated C/EBPβ protein isoform expression, associated with defective liver regeneration and impaired osteoclast differentiation. The high prevalence of uORFs in the human transcriptome suggests that intensified search for mutations within 5′ RNA leader regions may reveal a multitude of alterations affecting uORFs, causing pathogenic deregulation of protein expression.

Upstream ORFs (uORFs) are translational control elements found predominantly in transcripts of key regulatory genes. No mammalian genetic model exists to experimentally validate the physiological relevance of uORF-regulated translation initiation. We report that mice deficient for the CCAAT/enhancer-binding protein b (C/EBPb) uORF initiation codon fail to initiate translation of the autoantagonistic LIP (liver inhibitory protein) C/EBPb isoform. C/EBPb DuORF mice show hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. These data and the widespread prevalence of uORFs in mammalian transcriptomes suggest a comprehensive role of uORF-regulated translation in (patho)physiology.Supplemental material is available at http://www.genesdev.org.

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The balance between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to bone homeostasis, an equilibrium that is disturbed in many bone diseases. The transcription factor Tal1 is involved in the establishment of hematopoietic stem cells in the embryo and is a master regulator of hematopoietic gene expression in the adult. Here, we show that Tal1 is expressed in osteoclasts and that loss of Tal1 in osteoclast progenitors leads to altered expression of >1200 genes. We found that DC-STAMP, a key regulator of osteoclast cell fusion, is a direct target gene of Tal1 and show that Tal1 represses DC-STAMP expression by counteracting the activating function of the transcription factors PU.1 and MITF. The identification of Tal1 as a factor involved in cell fusion contributes to the understanding of osteoclast-associated diseases, including osteoporosis.

The CCAAT/enhancer binding proteins C/EBP␣ and C/EBP␤ are related transcription factors that are important for the function of various organs in the postnatal mouse. Gene replacement and tissue culture experiments have suggested partial redundancy of both transcription factors. Here we show that mouse embryos deficient of both C/EBP␣ and C/EBP␤ (C/EBP␣␤ ؊/؊ ) die between embryonic day 10 (E10) and E11 and display defective placentas. In situ hybridization revealed that C/EBP␣ and C/EBP␤ are coexpressed in the chorionic plate at E9.5 and later in the trophoblasts of the labyrinthine layer. In C/EBP␣␤ ؊/؊ placentas, allantoic blood vessels invaded the chorion; however, vessel expansion and development of the labyrinthine layer was impaired. Furthermore, a single copy of either C/EBP␣ in the absence of C/EBP␤ or C/EBP␤ in the absence of C/EBP␣ is sufficient to complete development, suggesting complementation of these C/EBPs during embryogenesis. A single copy of C/EBP␣ in the absence of C/EBP␤, however, fails to rescue survival after birth, suggesting haploinsufficiency of C/EBP␣ in newborns. Our data thus reveal novel essential, redundant, and dosage dependent functions of C/EBPs.

Lytic bone diseases and in particular osteoporosis are common age-related diseases characterized by enhanced bone fragility due to loss of bone density. Increasingly, osteoporosis poses a major global health-care problem due to the growth of the elderly population. Recently, it was found that the gene regulatory transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) is involved in bone metabolism. C/EBPβ occurs as different protein isoforms of variable amino terminal length, and regulation of the C/EBPβ isoform ratio balance was found to represent an important factor in osteoclast differentiation and bone homeostasis. Interestingly, adjustment of the C/EBPβ isoform ratio by the process of translational control is downstream of the mammalian target of rapamycin kinase (mTOR), a sensor of the nutritional status and a target of immunosuppressive and anticancer drugs. The findings imply that modulating the process of translational control of C/EBPβ isoform expression could represent a novel therapeutic approach in osteolytic bone diseases, including cancer and infection-induced bone loss.

BackgroundCell-based therapies such as autologous chondrocyte implantation are promising therapeutic approaches to treat cartilage defects to prevent further cartilage degeneration. To assure consistent quality of cell-based therapeutics, it is important to be able to predict the biological activity of such products. This requires the development of a potency assay, which assesses a characteristic of the cell transplant before implantation that can predict its cartilage regeneration capacity after implantation. In this study, an ex vivo human cartilage repair model was developed as quality assessment tool for potency and applied to co.don’s chondrosphere product, a matrix-associated autologous chondrocyte implant (chondrocyte spheroids) that is in clinical use in Germany.MethodsChondrocyte spheroids were generated from 14 donors, and implanted into a subchondral cartilage defect that was manually generated in human articular cartilage tissue. Implanted spheroids and cartilage tissue were co-cultured ex vivo for 12 weeks to allow regeneration processes to form new tissue within the cartilage defect. Before implantation, spheroid characteristics like glycosaminoglycan production and gene and protein expression of chondrogenic markers were assessed for each donor sample and compared to determine donor-dependent variation.ResultsAfter the co-cultivation, histological analyses showed the formation of repair tissue within the cartilage defect, which varied in amount for the different donors. In the repair tissue, aggrecan protein was expressed and extra-cellular matrix cartilage fibers were present, both indicative for a cartilage hyaline-like character of the repair tissue. The amount of formed repair tissue was used as a read-out for regeneration capacity and was correlated with the spheroid characteristics determined before implantation. A positive correlation was found between high level of aggrecan protein expression in spheroids before implantation and a higher regeneration potential after implantation, reflected by more newly formed repair tissue.ConclusionThis demonstrated that aggrecan protein expression levels in spheroids before implantation can potentially be used as surrogate potency assay for the cartilage cell transplant to predict its regenerative capacity after implantation in human patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1065-8) contains supplementary material, which is available to authorized users.