Citations

Delivery of biomolecules to the correct subcellular locales is critical for proper physiological function. To that end, we have previously determined that the type I and II TGFβ receptors (TβRI and TβRII, respectively) localize to the basolateral domain in polarized epithelia. While TβRII targeting was shown to be regulated by sequences between amino acids 529–538, the analogous region(s) within TβRI is unknown. To address that question, sequential cytoplasmic TβRI truncations and point mutations identified a targeting motif between residues 158 to 163 (VxxEED) required for basolateral TβRI expression. Further studies documented that receptor internalization, downregulation, direct recycling, or Smad signaling were unaffected by motif mutations which caused TβRI mislocalization. However, a 70 amino acid stretch containing the targeting motif was able to direct basolateral expression of the apically sorted Neurotrophin receptor (p75; extracellular and transmembrane regions) in a dominant manner. Lastly, co‐expression of apically targeted type I and type II TGFβ receptors mediated Smad3 signaling from the apical membrane of polarized epithelial cells. These later findings demonstrate that the absence of apical TGFβ signaling in normal epithelia reflects domain‐specific receptor expression and not an inability to couple with the signaling machinery. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.

The primary mediator of transforming growth factor β (TGFβ) responses are the Smad proteins. Here, we identify sorting nexin 9 (SNX9) as being required for Smad3 (but not Smad2) nuclear translocation and cell stimulation. SNX9 principally interacts with phosphorylated (p) Smad3 and promotes more rapid nuclear delivery from that observed independent of ligand. Although SNX9 does not bind Imp7, Impb, Nup153 or Nup214, it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates recruitment of pSmad3/SNX9 to the nuclear pore, heterodimerization of Imp8 with Impb, and nuclear translocation of pSmad3, but not SNX9. The demonstration that Smad3 is regulated in a distinct manner from Smad2 provides the opportunity to develop intervention strategies to enhance or dampen specific aspects of the cellular response to TGFβ. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.

Transforming growth factor‐β (TGFβ) is the prototype of a family of secreted polypeptide growth factors with key roles in cellular growth and differentiation. The primary mediators of TGFβ action are the Smad proteins, specifically Smad2 and Smad3. Upon receptor activation these 55–60 kDa proteins are phosphorylated (p) by the type I TGFβ receptor and undergo nuclear translocation to function as co‐modulators of gene expression. While the specific roles of Smad2 and Smad3 are being actively investigated, recent findings have shown that while Smad3 is clearly pro‐fibrotic/pro‐carcinogenic, Smad2 can induce anti‐fibrotic/anti‐carcinogenic responses. Thus, approaches designed to target more downstream TGFβ receptor signaling components may provide equivalent efficacy and greater specificity. To that end, as we previously determined that sorting nexin 9 (SNX9) plays an integral role in the nuclear import of pSmad3 (but not pSmad2), we have extended those findings and identified the functional element in SNX9 critical for pSmad3 binding. Furthermore, we show that a TAT‐fusion encompassing this region can act in a dominant manner to block SNX9/pSmad3 binding, pSmad3 nuclear translocation, and subsequent pro‐fibrotic responses dependent upon pSmad3 both in vitro and in vivo . Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.

Transforming growth factor beta (TGF‐β) plays a significant role in diverse biologic processes, including the regulation of cell growth, survival, differentiation, development, inflammation, immunity, hematopoiesis, and tissue remodeling. TGF‐β is essential for wound healing, stimulates matrix molecule deposition, and has been implicated in the pathogenesis of a variety of fibrotic disorders, including idiopathic pulmonary fibrosis, scleroderma, radiation‐induced pulmonary fibrosis, and asthma. Furthermore, activation of profibrotic TGF‐β signaling has a central role in glucose‐induced cell hypertrophy, induces glucose transporter 1, and regulates mitochondrial energetics. However, the mechanisms by which TGF‐β induces specific metabolic alterations in the fibrogenic process have not been fully defined. In this study, we demonstrate that TGF‐β stimulates type I TGF β receptor (TβRI) translocation to the mitochondria and induction of hexokinase 2 (HK2) via both Smad‐dependent and –independent pathways. Also, we showed that TbRI association with HK2 and the mitochondrial voltage‐dependent ion channel (VDAC). These findings suggest that TβRI plays a novel role in controlling mitochondrial function and regulation of glucose metabolism. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from the National Institutes of General Medical Sciences. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract In the current work we show that the profibrotic actions of TGF‐β are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF‐β required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF‐β. We further demonstrated that the NAD‐dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF‐β. Lastly, administration of the GLS1 inhibitor CB‐839 attenuated bleomycin‐induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF‐β‐dependent manner.

Abstract 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.

TGF-β–dependent metabolic dysregulation contributes to lung fibrosis.

Abstract Fibroproliferative diseases are a leading cause of morbidity and mortality featuring localized and systematic tissue/organ fibrosis, which account for nearly 45% of all deaths in the developed world, with an unknown etiology and no effective treatment. Transforming growth factor beta (TGF-β) is a ubiquitous cytokine that contributes to fibroblast activation, myofibroblast differentiation, collagen overproduction and pathological tissue fibrosis. In that mounting evidence indicate that altered glutamine metabolism has critical roles in regulating signaling pathways, maintaining redox balance and macromolecular biosynthesis, we wished to test the general hypothesis that intervention of these metabolic processes could provide novel approaches to developing anti-fibrotic therapies. To that end, in the current work we provide a framework integrating glutamine dysregulation with profibrotic TGF-β signaling. We found that Glutaminase 1 (GLS1), which converts glutamine to glutamate, is induced by TGF-β and is also elevated in (i) fibroblasts isolated from Idiopathic Pulmonary Fibrosis (IPF) patients; and (ii) the murine bleomycin (i.e., TGF-β driven) model of lung fibrosis. While phosphorylation of Smad2 and Smad3 occurs independently of GLS1, TGF-β stimulated expression of profibrotic targets (i.e., Collagen I, PAI-1, CTGF, α-SMA and fibronectin) as well as biological actions (i.e., cell migration and anchorage independent growth) require GLS1 activity. Moreover, knockdown of Smad2 or Smad3 as well as inhibition of MAPK, PI3K, Akt or mTOR abrogates the induction of GLS1 by TGF-β; documenting that TGF-β regulates GLS1 expression via both Smad and non-Smad signaling. As GLS1 represents a critical component mediating the fibroproliferative actions of TGF-β, in order to identify the operative molecular mechanism(s) we focused on (i) a group of histone deacetylases called sirtuins (SIRT) which are known to elicit antifibrotic effects; and (ii) the Forkhead box protein O4 (FOXO4) transcription factor which has been shown to bind the GLS1 promoter. We found that there is not only a decrease in SIRT7 and FOXO4 coincident with the induction of GLS1 by TGF-β, but TGF-β inhibits SIRT7 deacetylation activity which in turn prevents FOXO4 deacetylation and inhibition of GLS1 expression. Last, consistent with SIRT7 and FOXO4 being negative regulators of profibrotic TGF-β signaling, their levels were also decreased in IPF fibroblasts and bleomycin-induced lung fibrosis. These studies point to an exciting, novel and unexplored connection between sirtuins, FOXOs and glutamine metabolism by elucidating the mechanism(s) by which epigenetic and transcriptional processes cooperate to regulate glutamine metabolism and fibrotic development in a TGF-β dependent manner. Citation Format: Malay Choudhury, Xueqian Yin, Jeong-Han Kang, Mahefatiana Andrianifahanana, Edward B. Leof. Cooperativity between glutaminase 1 and histone deacetylase SIRT7 regulates TGF-β mediated lung fibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1847.

The subgroup A through E avian sarcoma and leukosis viruses ASLV(A) through ASLV(E) are a group of highly related alpharetroviruses that have evolved to use very different host protein families as receptors. We have exploited genetic selection strategies to force the replication-competent ASLVs to naturally evolve and acquire mutations to escape the pressure on virus entry and yield a functional replicating virus. In this study, evolutionary pressure was exerted on ASLV(B) virus entry and replication using a secreted for of its Tvb receptor. As expected, mutations in the ASLV(B) surface glycoprotein hypervariable regions were selected that knocked out the ability for the mutant glycoprotein to bind the sTvbS3-IgG inhibitor. However, the subgroup B Rous associated virus 2 (RAV-2) also required additional mutations in the C-terminal end of the SU glycoprotein and multiple regions of TM highlighting the importance of the entire viral envelope glycoprotein trimer structure to mediate the entry process efficiently. These mutations altered the normal two-step ASLV membrane fusion process to enable infection.

Evidence is provided that the fibroproliferative actions of TGF‐β are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF‐β signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF‐β‐stimulated FASN expression is independent of Smad2/ 3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF‐β ‐driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin‐treated mice, inhibition of FASN was examined in a murine‐treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.—Jung, M.‐Y., Kang, J.‐H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez‐Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF‐β signaling. FASEB J. 32, 3803–3815 (2018). www.fasebj.org