Yu-Hua Chow, Cecilia López-Martínez, W. Conrad Liles, William A. Altemeier, Sina A. Gharib, Chi F. Hung
Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip−/− mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip−/− mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip−/− mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, Tollip−/− mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip−/− MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip−/− MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip−/− MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip−/− through SMAD2 in vitro and in vivo. Tollip−/− mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip−/− mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.
{"title":"Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts","authors":"Yu-Hua Chow, Cecilia López-Martínez, W. Conrad Liles, William A. Altemeier, Sina A. Gharib, Chi F. Hung","doi":"10.1096/fba.2023-00054","DOIUrl":"10.1096/fba.2023-00054","url":null,"abstract":"<p>Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using <i>Tollip−/−</i> mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then <i>Tollip−/−</i> mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and <i>Tollip−/−</i> mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, <i>Tollip−/−</i> mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and <i>Tollip−/−</i> MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, <i>Tollip−/−</i> MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of <i>Acta2</i> by qPCR. Functionally, <i>Tollip−/−</i> MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in <i>Tollip−/−</i> through SMAD2 in vitro and in vivo. <i>Tollip−/−</i> mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, <i>Tollip−/−</i> mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.</p>","PeriodicalId":12093,"journal":{"name":"FASEB bioAdvances","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1096/fba.2023-00054","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139211308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Charles J. Kim, Chanpreet Singh, Marina Kaczmarek, Madison O'Donnell, Christine Lee, Kevin DiMagno, Melody W. Young, William Letsou, Raddy L. Ramos, Michael C. Granatosky, Michael Hadjiargyrou
Mustn1, a gene expressed exclusively in the musculoskeletal system, was shown in previous in vitro studies to be a key regulator of myogenic differentiation and myofusion. Other studies also showed Mustn1 expression associated with skeletal muscle development and hypertrophy. However, its specific role in skeletal muscle function remains unclear. This study sought to investigate the effects of Mustn1 in a conditional knockout (KO) mouse model in Pax7 positive skeletal muscle satellite cells. Specifically, we investigated the potential effects of Mustn1 on myogenic gene expression, grip strength, alterations in gait, ex vivo investigations of isolated skeletal muscle isometric contractions, and potential changes in the composition of muscle fiber types. Results indicate that Mustn1 KO mice did not present any substantial phenotypic changes or significant variations in genes related to myogenic differentiation and fusion. However, an approximately 10% decrease in overall grip strength was observed in the 2-month-old KO mice in comparison to the control wild type (WT), but this decrease was not significant when normalized by weight. KO mice also generated approximately 8% higher vertical force than WT at 4 months in the hindlimb. Ex vivo experiments revealed decreases in about 20 to 50% in skeletal muscle contractions and about 10%–20% fatigue in soleus of both 2- and 4-month-old KO mice, respectively. Lastly, immunofluorescent analyses showed a persistent increase of Type IIb fibers up to 15-fold in the KO mice while Type I fibers decreased about 20% and 30% at both 2 and 4 months, respectively. These findings suggest a potential adaptive or compensatory mechanism following Mustn1 loss, as well as hinting at an association between Mustn1 and muscle fiber typing. Collectively, Mustn1's complex roles in skeletal muscle physiology requires further research, particularly in terms of understanding the potential role of Mustn1 in muscle repair and regeneration, as well as with influence of exercise. Collectively, these will offer valuable insights into Mustn1's key biological functions and regulatory pathways.
Mustn1是一种只在肌肉骨骼系统中表达的基因,以前的体外研究表明它是肌原分化和肌融合的关键调节因子。其他研究也表明,Mustn1 的表达与骨骼肌的发育和肥大有关。然而,它在骨骼肌功能中的具体作用仍不清楚。本研究试图通过条件性基因敲除(KO)小鼠模型研究 Mustn1 对 Pax7 阳性骨骼肌卫星细胞的影响。具体来说,我们研究了Mustn1对肌源性基因表达、握力、步态改变、离体骨骼肌等长收缩的体内外调查以及肌肉纤维类型组成的潜在变化的潜在影响。结果表明,Mustn1 KO 小鼠没有出现任何实质性的表型变化,与肌原分化和融合相关的基因也没有显著变化。不过,与对照野生型(WT)相比,2 个月大的 KO 小鼠的总体握力下降了约 10%,但按体重归一化后,这种下降并不显著。4 个月大的 KO 小鼠后肢产生的垂直力也比 WT 小鼠高出约 8%。体内外实验显示,2 个月大和 4 个月大的 KO 小鼠骨骼肌收缩力分别下降了约 20% 至 50%,比目鱼肌疲劳力下降了约 10% 至 20%。最后,免疫荧光分析表明,KO 小鼠的 IIb 型纤维持续增加达 15 倍,而 I 型纤维在 2 个月和 4 个月时分别减少了约 20% 和 30%。这些发现表明,Mustn1缺失后可能存在一种适应或补偿机制,同时也暗示了Mustn1与肌肉纤维类型之间的关联。总之,Mustn1在骨骼肌生理学中的复杂作用需要进一步研究,尤其是在了解Mustn1在肌肉修复和再生中的潜在作用以及运动的影响方面。总之,这些研究将为了解 Mustn1 的关键生物学功能和调控途径提供有价值的见解。
{"title":"Mustn1 ablation in skeletal muscle results in functional alterations","authors":"Charles J. Kim, Chanpreet Singh, Marina Kaczmarek, Madison O'Donnell, Christine Lee, Kevin DiMagno, Melody W. Young, William Letsou, Raddy L. Ramos, Michael C. Granatosky, Michael Hadjiargyrou","doi":"10.1096/fba.2023-00082","DOIUrl":"https://doi.org/10.1096/fba.2023-00082","url":null,"abstract":"<p><i>Mustn1</i>, a gene expressed exclusively in the musculoskeletal system, was shown in previous in vitro studies to be a key regulator of myogenic differentiation and myofusion. Other studies also showed <i>Mustn1</i> expression associated with skeletal muscle development and hypertrophy. However, its specific role in skeletal muscle function remains unclear. This study sought to investigate the effects of <i>Mustn1</i> in a conditional knockout (KO) mouse model in Pax7 positive skeletal muscle satellite cells. Specifically, we investigated the potential effects of <i>Mustn1</i> on myogenic gene expression, grip strength, alterations in gait, ex vivo investigations of isolated skeletal muscle isometric contractions, and potential changes in the composition of muscle fiber types. Results indicate that <i>Mustn1</i> KO mice did not present any substantial phenotypic changes or significant variations in genes related to myogenic differentiation and fusion. However, an approximately 10% decrease in overall grip strength was observed in the 2-month-old KO mice in comparison to the control wild type (WT), but this decrease was not significant when normalized by weight. KO mice also generated approximately 8% higher vertical force than WT at 4 months in the hindlimb. Ex vivo experiments revealed decreases in about 20 to 50% in skeletal muscle contractions and about 10%–20% fatigue in soleus of both 2- and 4-month-old KO mice, respectively. Lastly, immunofluorescent analyses showed a persistent increase of Type IIb fibers up to 15-fold in the KO mice while Type I fibers decreased about 20% and 30% at both 2 and 4 months, respectively. These findings suggest a potential adaptive or compensatory mechanism following <i>Mustn1</i> loss, as well as hinting at an association between <i>Mustn1</i> and muscle fiber typing. Collectively, <i>Mustn1</i>'s complex roles in skeletal muscle physiology requires further research, particularly in terms of understanding the potential role of <i>Mustn1</i> in muscle repair and regeneration, as well as with influence of exercise. Collectively, these will offer valuable insights into <i>Mustn1</i>'s key biological functions and regulatory pathways.</p>","PeriodicalId":12093,"journal":{"name":"FASEB bioAdvances","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://faseb.onlinelibrary.wiley.com/doi/epdf/10.1096/fba.2023-00082","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138571072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Elena Silva, Véronique Ferchaud-Roucher, Anita Kramer, Lana Madi, Priyadarshini Pantham, Stephanie Chassen, Thomas Jansson, Theresa L. Powell
Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA-stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1-acylglycerol-3-phosphate-O-acyltransferase-4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.
胎儿的正常发育关键取决于胎盘的最佳营养供应,而胎盘氨基酸转运已被证明与胎儿的生长有积极的关系。雷帕霉素机制靶标(mTOR)是胎盘氨基酸转运体(如 A 系统)的正向调节因子。油酸(OA)先前已被证明对胎盘 mTOR 信号传导和原代人类滋养层细胞(PHT)的 A 系统氨基酸摄取有刺激作用。我们研究了 OA 与 PHT 中 A 系统活性之间的机理联系。我们发现,通过使用小干扰 RNA 敲除 raptor 或 rictor 来抑制 mTOR 复合物 1 或 2,可以阻止 OA 刺激的 System A 氨基酸转运,这表明 OA 与 mTOR 之间存在相互作用。磷脂酸(PA)是磷脂生物合成的关键中间体,也是mTOR通路的已知调节因子;然而,磷脂生物合成通路在胎盘中尚未得到广泛研究。我们发现了参与磷脂从头合成的胎盘酰基转移酶同工酶。沉默该途径中的一种酶--1-酰基甘油-3-磷酸-O-酰基转移酶-4,可阻止 OA 介导的 mTOR 刺激和 A 系统氨基酸转运。这些数据表明,OA 是通过 PA 的从头合成来刺激 PHT 细胞中的 mTOR 和氨基酸转运的。我们推测,母体循环中的脂肪酸(如 OA)通过与 mTOR 相互作用来调节对胎儿生长至关重要的胎盘功能,而妊娠晚期的高脂血症可能对增加胎儿的营养传输至关重要。
{"title":"Oleic acid stimulation of amino acid uptake in primary human trophoblast cells is mediated by phosphatidic acid and mTOR signaling","authors":"Elena Silva, Véronique Ferchaud-Roucher, Anita Kramer, Lana Madi, Priyadarshini Pantham, Stephanie Chassen, Thomas Jansson, Theresa L. Powell","doi":"10.1096/fba.2023-00113","DOIUrl":"10.1096/fba.2023-00113","url":null,"abstract":"<p>Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA-stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1-acylglycerol-3-phosphate-O-acyltransferase-4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.</p>","PeriodicalId":12093,"journal":{"name":"FASEB bioAdvances","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1096/fba.2023-00113","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}