Transforming growth factor β (TGF-β) is a central mediator of fibrogenesis. TGF-β is upregulated and activated in fibrotic diseases and modulates fibroblast phenotype and function, inducing myofibroblast transdifferentiation while promoting matrix preservation. Studies in a wide range of experimental models have demonstrated the involvement of the canonical ALK5/Smad3 pathway in fibrosis. Smad-independent pathways may regulate Smad activation and, under certain conditions, may directly transduce fibrogenic signals. The profibrotic actions of TGF-β are mediated, at least in part, through induction of its downstream effector, Connective Tissue Growth Factor. In light of its essential role in the pathogenesis of fibrosis, TGF-β has emerged as an attractive therapeutic target. However, the pleiotropic and multifunctional effects of TGF-β and its role in tissue homeostasis, immunity and cell proliferation raise concerns regarding potential side effects that may be caused by TGF-β blockade. This minireview summarizes the role of TGF-β signaling pathways in the fibrotic response.

SummaryDouble-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

The ability of DNA Double Strand Breaks (DSBs) to cluster in mammalian cells has been subjected to intense debate over the past few years. Here we used a high throughput chromosome conformation capture assay (Capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. We unambiguously found that DSBs do cluster but only when induced in transcriptionally active genes. Clustering of damaged genes mainly occurs during the G1 cell cycle phase and coincides with delayed repair. Moreover DSB clustering depends on the MRN complex, as well as the Formin 2 (FMN2) nuclear actin organizer and the LINC (LInker of Nuclear and Cytoplasmic skeleton) complex, suggesting that active mechanisms promote DSB clustering. This work reveals that when damaged, active genes exhibit a very peculiar behavior compared to the rest of the genome, being mostly left unrepaired and clustered in G1 while being repaired by homologous recombination in post-replicative cells.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

The matricellular protein Thrombospondin (TSP)-1 is induced following tissue injury and may regulate reparative responses by activating TGF-β, by suppressing angiogenesis, and by modulating inflammation and matrix metabolism. We hypothesized that endogenous TSP-1 may be involved in the pathogenesis of cardiac remodeling in the pressure-overloaded heart. Myocardial TSP-1 expression was increased in a mouse model of pressure overload due to transverse aortic constriction. TSP-1 -/- mice exhibited increased early hypertrophy and enhanced late dilation in response to pressure overload. Pressure-overloaded TSP-1 null mice had intense degenerative cardiomyocyte changes, exhibiting more extensive sarcomeric loss and sarcolemmal disruption when compared with wildtype hearts. Accentuated hypertrophy and cardiomyocyte injury in TSP-1 -/- hearts was accompanied by increased myofibroblast density. However, despite a two-fold higher infiltration of the cardiac interstitium with myofibroblasts, pressure-overloaded TSP-1 null hearts did not exhibit significantly increased collagen content when compared with wildtype hearts. The disproportionately low collagen content in TSP-1 null hearts was due to infiltration with abundant, but functionally defective, fibroblasts that exhibited impaired myofibroblast differentiation and reduced collagen expression in comparison to wildtype fibroblasts. Impaired myofibroblast activation in TSP-1 null hearts was associated with reduced Smad2 phosphorylation reflecting defective TGF-β signaling. Moreover, TSP-1 null hearts had increased myocardial Matrix Metalloproteinase (MMP)-3 expression and enhanced MMP-9 activation following pressure overload. TSP-1 upregulation in the pressure-overloaded heart critically regulates fibroblast phenotype and matrix remodeling by activating TGF-β signaling and by promoting matrix preservation, thus preventing chamber dilation.