Osteoarthritis is the most prevalent and crippling joint disease, and lacks curative treatment, as the underlying molecular basis is unclear. Here, we show that DOT1L, an enzyme involved in histone methylation, is a master protector of cartilage health. Loss of DOT1L disrupts the molecular signature of healthy chondrocytes in vitro and causes osteoarthritis in mice. Mechanistically, the protective function of DOT1L is attributable to inhibition of Wnt signalling, a pathway that when hyper-activated can lead to joint disease. Unexpectedly, DOT1L suppresses Wnt signalling by inhibiting the activity of sirtuin-1 (SIRT1), an important regulator of gene transcription. Inhibition of SIRT1 protects against osteoarthritis triggered by loss of DOT1L activity. Modulating the DOT1L network might therefore be a therapeutic approach to protect the cartilage against osteoarthritis.

BackgroundEffective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis.MethodsLung fibrosis was induced in wild-type or noggin haploinsufficient (Nog+/LacZ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis.ResultsNog+/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity.ConclusionsUpon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog+/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.

BackgroundThe pathogenesis of pulmonary fibrosis remains poorly understood. The Wnt signaling pathway regulates fibrogenesis in different organs. Here, we studied the role of two extracellular Wnt antagonists, secreted frizzled-related protein-1 (SFRP1) and frizzled-related protein (FRZB) on lung fibrosis in vitro and in vivo. For this purpose, we used an alveolar epithelial cell line and a lung fibroblast cell line, and the bleomycin-induced lung fibrosis model, respectively.ResultsDuring the course of bleomycin-induced lung fibrosis, Sfrp1 and Frzb expression are upregulated. Expression of Sfrp1 appears much higher than that of Frzb. In vitro, recombinant SFRP1, but not FRZB, counteracts the transforming growth factor β1 (TGFβ1)-induced upregulation of type I collagen expression both in pulmonary epithelial cells and fibroblasts. Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts, but not in alveolar epithelial cells. In vivo, Sfrp1 −/− and Frzb −/− mice showed identical responses to bleomycin in the lung compared to wild-type controls.ConclusionsAlthough SFRP1 counteracts the effect of TGFβ1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo. The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.

Background and objectives Fibrosis is the formation of excess fibrous connective tissue. It disturbs cell, tissue and organ homeostasis, leading to severe disease manifestations in various disorders. Transcriptional regulation of genes and pathways with pro- or anti-fibrotic effects is therefore a therapeutic target. DOT1-like histone H3 methyltransferase (DOT1L) is the only known H3K79 methyltransferase. It functions as a positive mediator of gene transcription, of DNA repair, as a regulator of the cell cycle, and has been associated with Wnt signalling. Our aim is to study the role of DOT1L in downstream events associated with pro-fibrotic cascades, hypothesising that it may act as an epigenetic masterswitch in the fibrotic process. Materials and methods MRC5 cells (a human fetal lung fibroblast cell line) were cultured for up to 22 days with 0 and 3µM of EPZ04777 (DOT1L inhibitor). During that period, the cells were stimulated at different time-ponts, with 0 to 5 ng/mL TGFβ1 for 24 h. We performed mRNA and protein expression analysis. Total RNA was extracted from cell lysates and reverse-transcribed, and gene expression levels were quantified with real-time quantitative PCR (qPCR). Expression was normalised to Hprt and subsequently normalised to the vehicle condition using the comparative cycle threshold method (ΔΔCT). Total cell lysates were homogenised in Cell Extraction Buffer and proteins were analysed by immunoblot, including assessment of the H3K79 methylation status. Results Long-term treatment progressively decreases the methylation status of H3K79. Upon stimulation with TGFβ1, MRC5 cells show upregulation of type I Collagen mRNA expression and collagen deposition. Moreover, αSMA mRNA expression is also increased, indicating a fibroblast to myofibroblast differentiation. TGFβ1 stimulation also increased DOT1L expression and activity in the absence of EPZ04777, as shown by qPCR and H3K79 methylation status. Upon treatment with EPZ04777, we also observed an increase in collagen and αSMA expression, suggesting that loss of DOT1L activity results in pro-fibrotic changes in lung fibroblasts. Conclusions DOT1L has homeostatic effects in the healthy lung fibroblast, since its inhibition triggers a pro-fibrotic transcriptional response. Its upregulation by TGFβ1 could represent a regulatory feedback loop that requires further investigation.