No abstract

Coordination of the cellular response to DNA damage is organised by multi-domain ‘scaffold’ proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.

Background: NF-B regulation of COL1A1 in physiopathological situations is largely unknown. Results: NF-B down-regulates COL1A1 in normal and scleroderma fibroblasts, through its recruitment on COL1A1 by protein interactions with the Sp1/Sp3/c-Krox trans-activators, which are different in fibrotic fibroblasts. Conclusion: Our findings highlight a new mechanism for COL1A1 regulation. Significance: These data could allow the development of new antifibrotic strategies.

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

A significant association between a polymorphism in the D repeat of the gene encoding asporin and osteoarthritis, the most frequent of articular diseases, has been recently reported. The goal of the present study was to investigate the expression of this new class I small leucine-rich proteoglycan (SLRP) in human articular chondrocytes. First, we studied the modulation of asporin (ASPN) expression by cytokines by Western blot and reverse transcription-polymerase chain reaction. Interleukin-1β and tumor necrosis factor-α downregulated ASPN, whereas transforming growth factor-β1 (when incubated in a serum-free medium) upregulated it. Similarly to proinflammatory cytokines, chondrocyte dedifferentiation induced by a successive passages of cells was accompanied by a decreased asporin expression, whereas their redifferentiation by three-dimensional culture restored its expression. Finally, we found an important role of the transcription factor Sp1 in the regulation of ASPN expression. Sp1 ectopic expression increased ASPN mRNA level and promoter activity. In addition, using gene reporter assay and electrophoretic mobility shift assay, we showed that Sp1 mediated its effect through a region located between -473 and -140 bp upstream of the transcription start site in ASPN gene. In conclusion, this report is the first study on the regulation of asporin expression by different cytokines in human articular chondrocytes. Our data indicate that the expression of this gene is finely regulated in cartilage and suggest a major role of Sp1.

Long-term cultures under hypoxic conditions have been demonstrated to maintain the phenotype of mesenchymal stromal/stem cells (MSCs) and to prevent the emergence of senescence. According to several studies, hypoxia has frequently been reported to drive genomic instability in cancer cells and in MSCs by hindering the DNA damage response and DNA repair. Thus, we evaluated the occurrence of DNA damage and repair events during the ex vivo expansion of clinical-grade adipose-derived stromal cells (ADSCs) and bone marrow (BM)-derived MSCs cultured with platelet lysate under 21% (normoxia) or 1% (hypoxia) O 2 conditions. Hypoxia did not impair cell survival after DNA damage, regardless of MSC origin. However, ADSCs, unlike BMMSCs, displayed altered cH2AX signaling and increased ubiquitylated cH2AX levels under hypoxic conditions, indicating an impaired resolution of DNA damage-induced foci. Moreover, hypoxia specifically promoted BM-MSC DNA integrity, with increased Ku80, TP53BP1, BRCA1, and RAD51 expression levels and more efficient nonhomologous end joining and homologous recombination repair. We further observed that hypoxia favored mtDNA stability and maintenance of differentiation potential after genotoxic stress. We conclude that long-term cultures under 1% O 2 were more suitable for BM-MSCs as suggested by improved genomic stability compared with ADSCs. STEM CELLS 2015;33:3608-3620 SIGNIFICANCE STATEMENTHuman mesenchymal stem cells (MSCs) feature differentiation capacities, immunomodulatory properties and therefore represent a great potential for medicine. In vitro culture allows their amplification for suitable clinical uses. Hypoxia seems relevant for the prevention of senescence and loss of MSC features. However, hypoxia is shown to promote cancer or DNA damage response (DDR) and repair failures. We demonstrate that long-term culture of MSCs with platelet lysate in hypoxia does not necessarily inhibit DDR and DNA repair. Here, bone marrowMSCs conserved or rather improved their DDR and DNA repair (NHEJ, HR), mtDNA stability in hypoxia compared to adipose-derived stem cells. Moreover, the ubiquitylation status of gH2AX between BM-MSCs and ADSCs might be responsible for changes in their DNA repair activities. Appropriate oxygen tension seems crucial to preserve the MSCs genomic stability according to their origins.

Type II collagen is a marker of articular cartilage encoded by the COL2A1 gene. The nature of the trans factors involved in the upregulation of this gene by insulin-like growth factor-I (IGF-I) remains unclear. We found that IGF-I increased type II collagen synthesis by a transcriptional control mechanism involving a 715-bp region within the COL2A1 first-intron specific enhancer. The overproduction of L-Sox5/Sox6/Sox9 and Sp1 and decoy experiments targeting these factors demonstrated their action in concert in IGF-I trans-activation. These results were supported by the data obtained in knockdown experiments in which siRNA against Sox9/Sox6 and Sp1 prevented the IGF-I-induced increase in collagen II production. Indeed, each of these trans-activators increased the expression of others. IGF-I increased the binding of Sox9 and Sp1/Sp3 to their cis elements in the enhancer, and we provide the first evidence of Sox9 interaction with the promoter by chromatin immunoprecipitation. Interactions with COL2A1 were also observed for Sp1, p300/CBP, and Tip60. Finally, a physical interaction between Sox9, p300, Sp3, and Sp1 was detected. These data demonstrate the role of Sox9, Sp1/Sp3, and euchromatin-associated factors (p300, Tip60) in the IGF-I-induced upregulation of COL2A1, indicating possible use of this growth factor in articular cartilage engineering applications to promote repair in patients with degenerative diseases, such as osteoarthritis.

The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17-estradiol (17-E 2 ) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17-E 2 stimulates, via its receptor hER66 (human estrogen receptor  66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand independent transactivation domain (AF-1) of hER66 was mediated by 'GC' binding sites of the -266/-63 bp promoter, through physical interactions between ER, Sp1/Sp3, Sox9 and p300, as demonstrated in ChIP and Re-ChIP assays in primary and dedifferentiated cells. 17-E 2 and hER66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3 and Sox-9 siRNAs prevented hER66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hER66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17-E 2 and hER66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17-E 2 could promote chondrocyte redifferentiation. Keywords Key Messages 17-E 2 up-regulates type II collagen gene expression in articular chondrocytes.  An ER66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect.  This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo.  Our findings highlight a new regulatory mechanism for 17-E 2 action in chondrocytes.  17-E 2 might be an attractive candidate for cartilage engineering applications.2