Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/β-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein–1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O₂between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O₂, and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-β type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-β signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O₂as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O₂. After exposure to 85% O₂, primary alveolar type II cells were more susceptible to TGF-β-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O₂significantly enhanced the TGF-β-stimulated production of the α₁subunit of type I collagen (Iα₁), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-β/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N⁶-methyladenosine (m⁶A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m⁶A in 18S rRNA at position A₁₈₃₂. We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m⁶A in rRNA in stemness, differentiation, development, and diseases.

Dietary restriction regimes extend lifespan in various animal models. Here we show that longevity in male C57BL/6J mice subjected to every-other-day feeding is associated with a delayed onset of neoplastic disease that naturally limits lifespan in these animals. We compare more than 200 phenotypes in over 20 tissues in aged animals fed with a lifelong every-other-day feeding or ad libitum access to food diet to determine whether molecular, cellular, physiological and histopathological aging features develop more slowly in every-other-day feeding mice than in controls. We also analyze the effects of every-other-day feeding on young mice on shorter-term every-other-day feeding or ad libitum to account for possible aging-independent restriction effects. Our large-scale analysis reveals overall only limited evidence for a retardation of the aging rate in every-other-day feeding mice. The data indicate that every-other-day feeding-induced longevity is sufficiently explained by delays in life-limiting neoplastic disorders and is not associated with a more general slowing of the aging process in mice.

Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disease unresponsive to currently available therapies. In IPF, initial alveolar epithelial cell damage leads to activation of fibroblast-(myo)fibroblasts, which deposit an increased amount of a collagen-rich extracellular matrix. Angiotensin II (ANGII) signaling, mediated via angiotensin II receptor type 1 (AGTR1) or type 2 (AGTR2), controls tissue remodeling in fibrosis, but the relevance of AGTR2 remains elusive. In the present study, we demonstrated increased expression of AGTR1 und AGTR2 in human and rodent lung tissues from patients with IPF and mice subjected to bleomycin-induced fibrosis, respectively. Both AGTR1 und AGTR2 localized to interstitial fibroblasts. Quantitative analysis of cell surface expression in primary mouse fibroblasts revealed a significant increase of AGTR2 surface expression in fibrotic fibroblasts, whereas AGTR1 surface expression levels remained similar. ANGII treatment of normal fibroblasts led to enhanced migration and proliferation, which was abrogated after pretreatment with losartan (LOS), an AGTR1 inhibitor. In contrast, in fibrotic fibroblasts, migration and proliferation was modified only by AGTR2, but not AGTR1 inhibition (using PD123319). ANGII-induced effects were mediated via phosphorylation of the mitogen-activated protein kinases p38 and p42/44, which was blocked via LOS and PD123319, respectively. Similar effects of AGTR1 and AGTR2 inhibition were observed using conditioned media of alveolar epithelial cells, a prominent source of ANGII in the lung in vivo. In summary, we conclude that ANGII signaling occurs primarily via AGTR1 in normal fibroblasts, while AGTR2-mediated effects are dominant on activated (myo)-fibroblasts, a receptor switch that may perturb epithelial–mesenchymal interaction, thereby further perpetuating fibrogenesis.

Background Idiopathic pulmonary fibrosis (IPF) has a poor prognosis and limited responsiveness to available treatments. It is characterised by epithelial cell injury, fibroblast activation and proliferation and extracellular matrix deposition. Serotonin (5-hydroxytryptamine; 5-HT) induces fibroblast proliferation via the 5-HTR_2A and 5-HTR_2B receptors, but its pathophysiological role in IPF remains unclear. A study was undertaken to determine the expression of 5-HT receptors in IPF and experimental lung fibrosis and to investigate the effects of therapeutic inhibition of 5-HTR_2A/B signalling on lung fibrosis in vivo and in vitro. Methods and results Quantitative RT-PCR showed that the expression of 5-HTR_1A/B and 5-HTR_2B was significantly increased in the lungs of patients with IPF (n=12) and in those with non-specific interstitial pneumonia (NSIP, n=6) compared with transplant donors (n=12). The expression of 5-HTR_2A was increased specifically in IPF lungs but not in NSIP lungs. While 5-HTR_2A protein largely localised to fibroblasts, 5-HTR_2B localised to the epithelium. To assess the effects of 5HTR_2A/B inhibition on fibrogenesis in vivo, mice were subjected to bleomycin-induced lung fibrosis and treated with the 5-HTR_2A/B antagonist terguride (or vehicle) in a therapeutic approach (days 14–28 after bleomycin). Terguride-treated mice had significantly improved lung function and histology and decreased collagen content compared with vehicle-treated mice. Functional in vitro studies showed that terguride is a potent inhibitor of transforming growth factor β₁- or WNT3a-induced collagen production. Conclusion The studies revealed an increased expression of 5-HTR_2A specifically in IPF. Blockade of 5-HTR_2A/B signalling by terguride reversed lung fibrosis and is thus a promising therapeutic approach for IPF.