Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor–β (TGF-β). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-β both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-β signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-β–stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-β signaling.

Transforming growth factor-beta (TGFβ) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFβ is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFβ induces the expression of the immunoinhibitory molecule Programed death-ligand 1 (PD-L1) in human and murine fibroblasts in a Smad2/3-and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFβ-dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFβ signaling, TGFβ stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases. K E Y W O R D Sfibroblast, checkpoint inhibitor, extracellular vesicles, signaling 2214 | KANG et Al. | 2223 KANG et Al. F I G U R E 6EVs from TGFβ treated fibroblasts inhibit T cell proliferation and cell migration. A, MRC5 cultures were treated in the absence(−) or presence (+) of TGFβ (10 ng/mL) for 3 days. Following the collection of the supernatant for EV isolation, the cells were lysed, and both were assessed for expression of PD-L1 or the EV-associated protein CD63 by Western blotting (representative of 3 independent experiments). B, EV particle size distribution was identified through nanoparticle tracking analysis and processed with NTA software (NanoSight). C, EVs were purified from MRC5 cells cultured in the absence (−) or presence (+) of TGFβ as in (A). Peripheral blood mononuclear cells (n = 5 volunteers) were then treated with anti-CD3 (+αCD3; 500 ng/mL) to activate T cell proliferation and incubated for 6 days with no EVs, EVs (10 μg/mL) isolated from cells ± TGFβ, or EVs isolated from cells ± TGFβ ± blocking antibody to PD-L1 (10 μg/mL Avelumab). The percent change in proliferation, measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) content, compared to anti-CD3 treatment alone is shown. D, (Top 2 panels) Transwell invasion assays were performed in MRC5 cells treated in 0.1% FBS/DME alone (Con) or supplemented with 5 ng/mL TGFβ for 18 hours as described in Materials and Methods. (Bottom 3 panels) MRC5 cells were assessed as in the top panels except that the 0.1% FBS/DME contained EVs (10 μg/mL) isolated from MRC5 cells ± TGFβ or MRC5 cells which had been treated with siRNA to PD-L1 and then stimulated with TGFβ. E, Quantitation of transwell migration of cells treated in (D). Data reflect mean ±...

Reb1 of Schizosaccharomyces pombe represents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4-14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction. Several studies have suggested that pol I transcription termination is a multistep process that requires (i) pausing of chain elongation by the terminator protein and (ii) dissociation and release of pol I and the primary transcript from the template. In mice, dissociation of pol I and release of the transcript require the release factor PTRF, a 44-kDa protein that interacts with the largest subunit of pol I (15). In yeast, additional factors for the processing of the end include the endonuclease Rnt1, the 5′-3′ Rat1 exonuclease, Sen1 helicase, and the kinase Grc3. They are part of an alternate pathway for termination by transcriptional coprocessing (4,(16)(17)(18)(19). In addition, in vivo analyses of Saccharomyces cerevisiae have shown a requirement for Rpa12, a component of pol I necessary for transcription termination (20). Whether the polar arrest of transcription is caused by an interaction between Rpa12 with the terminator protein is unknown.The terminator proteins that mediate transcription termination have been identified in multiple organisms and include Reb1 and Nsi1 [also called yeast transcription terminator (Ytt1)] of S. cerevisiae (...

Pathogenic fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two‐thirds of IPF patients die within 2‐5 years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFβ) and insulin‐like growth factor‐1 (IGF‐1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF‐1 in the initiation and progression of TGFβ‐induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF‐1 via TGFβ in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF‐1's C1 promoter as mediating the increase in IGF‐1 transcription by TGFβ in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFβ‐dependent Igf‐1 expression in myofibroblasts; (4) demonstrates IGF‐1R activation is essential to support TGFβ‐driven profibrotic myofibroblast functions and excessive wound healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF‐1R inhibitor OSI‐906. These findings expand our knowledge of IGF‐1's role as a novel fibrotic‐switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.