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. 2025 Feb 25;73(3):441–450. doi: 10.1165/rcmb.2024-0263OC

The Role of Tuftelin-1 in Mesomesenchymal Transition of Pleural Mesothelial Cells and the Progression of Pleural Fibrosis

Ann Jeffers 1,2, Shuzi Owens 2, Wenyi Qin 2, Olamipejo Durojaye 2, Matt Florence 2, Peace Okeke 2, Luis Destarac 3, Shiva Keshava 2, Mitsuo Ikebe 2, Steven Idell 1,2, Torry A Tucker 1,2,✉
PMCID: PMC12416313  PMID: 39998837

Abstract

Pleural conditions causing exudative effusions (empyema or complicated parapneumonia) can result in pathological pleural organization leading to pleural fibrosis (PF). Pleural mesothelial cells (PMCs) undergo mesenchymal transition (MesoMT) and acquire a profibrotic phenotype characterized by increased expression of ACTA2; collagen type I (Col-1); and phenotypic changes, including elongation, stress fiber formation, and contraction. Using RNA-sequencing analysis, we identified Tuftelin-1 (Tuft1) as a novel potential target. Although prior studies have shown that Tuft1 expression is associated with aggressive cellular phenotypes, its role in PF is unknown. Our prior studies show that inhibition of PI3K/Akt, mTORC2, or GSK-3β blocks MesoMT. In this study, we build on previous findings and suggest that Tuft1 plays a key role in promoting MesoMT. In human PMCs, various mediators that induce MesoMT result in upregulation of Tuft1 expression. Furthermore, we also found that Tuft1 was increased in human pleuritis tissues and in murine models of PF compared with normal lung. In our studies, TGF-β–mediated increase in Tuft1 was blocked by the GSK-3β inhibitor 9-ING-41. Knockdown of Tuft1 in vitro blocked TGF-β–mediated MesoMT. Conversely, Tuft1 overexpression induced mTORC2 signaling and promoted MesoMT in the absence of TGF-β. In vivo analyses showed that mesothelial cell–specific Tuft1 knockout mice (Tuft1PMC−/−) were protected from Streptococcus pneumoniae–mediated pleural injury. Histological analysis showed that pleural thickening and profibrotic markers were significantly reduced in Tuft1PMC−/− mice compared with wild-type control animals. These studies strongly support therapeutic targeting of Tuft1 as a novel means to mitigate PF.

Keywords: pleural mesothelial cells, transforming growth factor-β, Tuftelin-1, mTORC2

Clinical Relevance

Our research identifies Tuftelin-1 as a novel therapeutic target for preventing the progression of pleural fibrosis. For the first time, to our knowledge, we demonstrate that Tuftelin-1 plays a crucial role in the mesenchymal transition of mesothelial cells induced by multiple mediators. Moreover, our findings contribute new insights to studies on TGF-β–induced signaling pathways.

Pleural conditions such as empyema and complicated parapneumonic effusion can lead to fibrosing pleural lung disease preceded by significant extravascular fibrin deposition and its organization. This process occurs within the visceral and parietal pleura leading to pleural fibrosis (PF) (1, 2). In fibrosing pleural disease, myofibroblasts accumulate in the thickened pleural rind driving PF. These myofibroblasts are known to promote disease progression via the increased expression of extracellular matrix proteins, including collagen and fibronectin. We and others have reported that resident pleural mesothelial cells contribute to this population via a process termed “mesomesenchymal transition” (MesoMT) (3–7). However, the mechanism(s) that govern their appearance and persistence have not been fully elucidated.

A diverse array of signaling mediators contribute to the progression of MesoMT, including transforming growth factor-β (TGF-β)-mediated activation of PI3K (phosphoinositide 3-kinase)/Akt and GSK-3β (glycogen synthase kinase-3β) (8, 9). Recently, we reported that mechanistic/mammalian target of rapamycin complex 2 (mTORC2) plays a significant role in the induction of MesoMT and the progression of PF (3). This complex contains the rapamycin-insensitive companion of mTOR (RICTOR) and other associated proteins. Activation of mTORC2 can increase activation of Akt signaling by phosphorylating Akt at serine 473 (Ser473). This mechanism allows mTORC2 to potentiate signaling of the mTORC1 complex. Unlike mTORC1, a clear understanding of mTORC2, its regulation, activation, and related signaling has been limited.

Tuftelin 1 (Tuft1) is an acidic glycoprotein reported to play a role in the mineralization of tooth enamel. It is a highly conserved gene that is highly homologous across species (10–12). However, Tuft1 is also expressed in hypoxic conditions, such as cancer (10, 13–15). Tuft1 expression negatively correlates with survival in diverse cancers, including triple-negative breast cancer, thyroid carcinoma, lung adenocarcinoma, and pancreatic cancer (10, 16–18). Tuft1 expression did not correlate to lung fibrosis in publicly available databases. However, Tuft1 downregulation was reported to protect against fibrosing lung injury in a mouse model (19). Recently, Tuft1 expression was reported to regulate tumor cell resistance to chemotherapy via regulation of mTORC1 signaling (10). However, the definitive mechanism of action of Tuft1 remains under intense investigation. Here we report, for the first time, to our knowledge, that Tuft1 expression is critical for mTORC2 activation and the progression of MesoMT in vitro and in vivo.

Methods

Primary Pleural Mesothelial Cell Isolation and Culture

Human pleural mesothelial cells (HPMCs) were isolated from pleural fluids collected from patients with congestive heart failure or post–coronary bypass pleural effusions, as previously described (20). HPMCs were cultured in a humidified incubator at 37°C in 5% CO2 and 95% air. Cells were maintained in bronchial epithelial cell growth medium (containing the BulletKit minus epinephrine and retinoic acid; Lonza) containing 3% FBS (Gibco), 2% antibiotic-antimycotic (Lonza), and 1% GlutaMAX (Invitrogen), as previously described (3–5). Primary HPMCs were passed a maximum of five times. Only cells with a calretinin positivity >85% were used for the indicated studies.

Treatment with Mediators of MesoMT and Sample Collection

HPMCs were first serum starved in RPMI 1640 media containing GlutaMAX, as previously reported (3–5, 21). Cells were then treated with TGF-β (5 ng/ml), factor Xa (FXa; 7 nM), thrombin (13 nM), and plasmin or urokinase plasminogen activator (uPA; 20 nM) for 24 hours for RNA analyses. RNA was then collected using a Qiagen RNA isolation kit according to the manufacturer’s instructions. Isolated RNA was then transcribed into cDNA (Bio-Rad reverse transcriptase kit) and analyzed by qPCR analyses using the CFX Bio-Rad Opus PCR Machine (see Table E1 in the data supplement).

For protein analyses, serum-starved cells were treated for 48 hours and then lysed using PBX-100 containing protease and phosphatase inhibitors (Sigma-Aldrich), as previously reported (3, 8, 21, 22). Collected lysates were cleared via centrifugation and protein quantified using the bicinchoninic acid assay (Thermo Fisher). Lysates were then resolved via SDS-PAGE and transferred to polyvinylidene fluoride membrane for antibody probing analyses.

Streptococcus pneumoniae–mediated Pleural Injury Model of PF

S. pneumoniae–mediated pleural injury was initiated to induce PF as previously described (3, 4, 21, 23). Pan-Tuft1–deficient mice die within hours of birth (10). We therefore generated conditional, mesothelial cell–specific Tuft1-knockout mice (Tuft1PMC−/−) by crossing floxed Tuftelin 1 mice (generated via contract with The Jackson Laboratory) with calb2-cre mice (Calb2-IRES-Cre, The Jackson Laboratory, 010774). Wild-type (WT) C57BL/6J mice (The Jackson Laboratory) and Tuft1PMC−/− mice were intrapleurally administered 1.8 × 108 cfu of S. pneumoniae. Antibiotic treatment (ampicillin 100 mg/kg) was administered via intraperitoneal injection beginning at 4 hours after inoculation, followed by three more doses each at 24-hour intervals. A total of four doses of antibiotics were administered. After 7 days, mice were killed via exsanguination. Lungs were then inflated and collected in a fixative solution for further analysis.

Results

Mediators of MesoMT Induce Tuft1 Expression

Because Tuft1 was identified as being upregulated in cells undergoing MesoMT (data not shown), we examined if Tuft1 expression is increased in human pleuritis lung samples. We previously reported that tissue sections from the lungs of patients with pleuritis demonstrated elevated expression of MesoMT markers, such as ACTA2 and collagen type I (3, 4, 21, 24). We also found that pleural mesothelial cells in the same sections costained for the mesothelial cell marker calretinin. Tissue sections from normal and pleuritis lungs were stained for Tuft1 (Figure 1A). Although Tuft1 expression was detectable in normal human lung tissue sections, enhanced Tuft1 expression was found at the pleural surface of lung sections from patients with pleuritis. We also found that enhanced Tuft1 expression colocalized with calretinin-positive pleural mesothelial cells in human pleuritis tissue sections (Figure E1). Similar results were observed in lung tissues from mice in the S. pneumoniae model of PF (Figure 1B). However, Tuft1 expression was below the level of detection in saline-treated mice. These data suggest that Tuft1 plays a role in the progression of MesoMT. We previously showed that diverse mediators induce MesoMT of PMCs (20). This list includes TGF-β, FXa, thrombin, plasmin, and uPA. Furthermore, we reported that these same mediators activate mTORC2 signaling, which is critical for the induction of PF (3). Accordingly, we next evaluated the effect of these mediators on the expression of Tuft1 in HPMCs. HPMCs were treated with TGF-β, FXa, thrombin, plasmin, and uPA. HPMC lysates were then probed for Tuft1. As expected, these mediators induced MesoMT, as indicated by increased ACTA2 expression (Figure 2). These mediators also activated the mTORC2 signaling pathway, as demonstrated by phosphorylation of Akt at Ser473 (Figure 2). Tuft1 expression was significantly induced by TGF-β and thrombin (P < 0.05). Although uPA did not affect Tuft1 expression, increases in Tuft1 by treatment with FXa and plasmin approached significance (P < 0.08 and 0.09, respectively).

Figure 1.

Figure 1.

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Tuftelin-1 (Tuft1) expression is increased in the pleural mesothelium of pleuritic lung tissues. (A) Normal and nonspecific pleuritis human lung tissues were sectioned and immunostained for Tuft1 (red) and nuclei (blue). Increased Tuft1 expression was observed at the surface of the pleural mesothelium in pleuritis sections compared with normal tissue samples. Scale bars, 100 μm. (B) Lung tissue sections from normal and Streptococcus pneumoniae (Strep) injured mice (7 d) were stained for Tuft1 (red) and cell nuclei (blue). Increased Tuft1 expression was observed at the surface of the pleural mesothelium in S. pneumoniae injured mice. Images are representative of n = 3/group. Scale bars, 50 μm.

Figure 2.

Figure 2.

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Mesenchymal transition (MesoMT) mediators induce Tuft1 expression. Serum-starved HPMCs were treated with TGF-β (5 ng/ml), factor Xa (FXa; 7 nM), THB (13 nM), PLN (6 nM), or uPA (20 nM) for 48 hours. Cell lysates were then observed for changes in the expression of ACTA2 and Tuft1. The mechanistic/mammalian target of rapamycin complex 2 (mTORC2) signaling intermediate Akt (Ser473) was also probed. β-Actin was used as the loading control. The graphic depicts the densitometric measurements of Tuft1 expression in treated HPMCs. *P < 0.05 and **P < 0.01. Images are representative of four independent experiments. HPMCs = human pleural mesothelial cells; PLN = plasmin; THB = thrombin; uPA = urokinase plasminogen activator.

Tuft1 Downregulation Blocks Induction of MesoMT

Because multiple mediators of MesoMT significantly induced Tuft1 expression, we next evaluated the effect of Tuft1 downregulation on induction of MesoMT. Untransfected, control siRNA and Tuft1 siRNA-transfected HPMCs were treated in the presence or absence of TGF-β. We first confirmed whether Tuft1 siRNA could reduce TGF-β–mediated increases in Tuft1 mRNA expression. Tuft1 siRNA significantly reduced TGF-β–mediated increases in Tuft1 expression (Figure 3A; P < 0.01) to levels below baseline PBS levels. As we and others have previously reported, TGF-β increased ACTA2 and collagen type I (Col1) mRNA expression in HPMCs (7, 25, 26). Conversely, Tuft1 downregulation significantly reduced TGF-β–induced ACTA2 and Col1 expression compared with similarly treated untransfected and control siRNA cells (Figure 3B). Akt phosphorylation at Ser473 was likewise reduced in Tuft1 downregulated HPMCs. Similar results were observed in FXa-treated (Figure 3C) and thrombin-treated (Figure 3D) HPMCs, because Tuft1 knockdown cells demonstrated reduced induction of MesoMT compared with untransfected and control transfected cells.

Figure 3.

Figure 3.

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Tuft1 expression mediates the progression of MesoMT. HPMCs were untransfected or transfected with control (siCont) or Tuft1 (siTuft1)-specific siRNAs. Cells were then serum starved for 12–16 hours and treated with TGF-β (5 ng/ml) for 24 hours (qPCR; A) or 48 hours (Western blot analysis; B). RNA was isolated 24 hours after treatment (A) and converted to cDNA. Tuft1 downregulation was confirmed via qPCR analyses. Changes in ACTA2 and collagen type I (Col1) mRNA expression levels were also analyzed by qPCR. (B) Cell lysates from treated cells were obtained 48 hours after stimulation with TGF-β, immunoblotted, and probed for ACTA2 and Tuft1 expression. AKT and β-actin were used as loading controls. Equal volumes of the conditioned media were probed for Col1 protein. TGF-β–mediated increases in ACTA2 and Col1 were significantly reduced in Tuft1 downregulated cells but not in the untransfected and control siRNA HPMCs. Untransfected, control siRNA and Tuft1 siRNA transfected cells were treated with FXa (C) and thrombin (D) for 48 hours. Cell lysates were then isolated and probed for changes in Tuft1 and ACTA2. FXa- and thrombin-mediated changes in ACTA2 were blocked in Tuft1 downregulation. Data are representative of a minimum of three independent experiments and are presented as mean ± SEM and evaluated by one-way ANOVA with Dunnett post hoc test. For all studies, *P < 0.05, **P < 0.01, and ***P < 0.001. At least two reference genes were used to calculate the relative expression levels of target genes in qPCR studies.

Conversely, Tuft1 overexpression via adenoviral transduction significantly induced Akt phosphorylation at Ser473 compared with TGF-β–treated control and GFP-expressing HPMCs (Figure 4), suggesting potent activation of the mTORC2 pathway. GFP adenovirus treatment also significantly increased Ser473 phosphorylation compared with uninfected HPMCs. However, Tuft1 expressing HPMCs showed significantly higher Akt phosphorylation (Ser473) compared with similarly treated GFP-expressing HPMCs in the presence and absence of TGF-β. Akt-Thr308 and S6-Ser240 phosphorylation were also increased in Tuft1-expressing HPMCs. These phosphorylation events show that the mTORC1 pathway is likewise activated by enhanced Tuft1 expression.

Figure 4.

Figure 4.

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Tuft1 expression induces mTORC1 and mTORC2 activation. HPMCs were either uninfected, infected with GFP adenovirus (AdV-GFP), or infected with Tuft1 adenovirus (AdV-Tuft1). Serum-starved HPMCs were then treated with TGF-β (5 ng/ml) for 48 hours. Cell lysates were probed for Tuft1, ACTA2, and phosphorylated Akt (Ser473 and Thr308). β-Actin was the loading control. Densitometric analyses were performed on Akt Ser473 phosphorylation, and values were normalized to PBS (100%) and graphed. Akt phosphorylation at Ser473 was significantly increased by Tuft1 expression. All data are representative of four independent experiments and are presented as mean ± SEM. Data were evaluated by one-way ANOVA with Dunnett post hoc test. For all studies, *P < 0.05 and **P < 0.01.

Tuft1 Expression Is GSK-3β but Not mTORC1/2 Dependent

Prior work showed that GSK-3β contributes to the progression of MesoMT (8, 27). Furthermore, mTORC2 signaling in HPMCs was muted by GSK-3β inhibition with 9-ING-41 (3). Because of these findings, we next determined the effect of GSK-3β inhibition on Tuft1 expression (Figure 5A). Total RNA was isolated from TGF-β–treated cells in the presence and absence of varying concentrations of 9-ING-41 (10–0.5 μM) and analyzed by qPCR. Pretreatment of HPMCs with varying doses of 9-ING-41 followed by TGF-β treatment significantly blunted the induction of ACTA2 mRNA at the highest doses (10, 5, and 1 μM) compared with TGF-β alone (P < 0.05; Figure 5A). Similar results were observed for Tuft1 expression, because TGF-β–mediated increases in Tuft1 were significantly blocked by 10 and 5 μg/ml doses of 9-ING-41. These findings were confirmed using a second GSK-3β inhibitor, TDZD-8 (8), because the highest dose (30 μM) significantly reduced Tuft1 induction by TGF-β (Figure E2). The role of mTORC1/2 signaling in Tuft1 expression was next determined using the mTORC1/2 inhibitor INK128 (Figure 5B). Unlike 9-ING-41, mTORC1/2 inhibition, using INK128 (3), did not affect Tuft1 expression. Rather, INK128 significantly increased TGF-β–mediated changes in Tuft1 mRNA (P < 0.05; 0.025 and 0.01 μM INK128). Similar results were observed with the SMAD3 inhibitor, SIS3 (data not shown), because TGF-β–mediated increases in Tuft1 were unaffected by SMAD3 inhibition.

Figure 5.

Figure 5.

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Glycogen synthase kinase-3β (GSK-3β) inhibition blocks induction of Tuft1. HPMCs were treated with 9-ING-41 (A) or INK128 (B) before treatment with TGF-β. After 24 hours of TGF-β treatment, RNA was isolated, converted to cDNA, and probed for ACTA2 and/or Tuft1. (A) Decreasing doses of the GSK-3β inhibitor 9-ING-41 significantly blocked the induction of MesoMT (ACTA2) and TGF-β–mediated induction of Tuft1. (B) Decreasing doses of INK128 significantly increased TGF-β–mediated increases in Tuft1. Graphs represent n = 3–4 independent experiments. Data were evaluated by one-way ANOVA with Dunnett post hoc test. For all studies, *P < 0.05. GAPDH was used as the reference gene.

Conditional Tuft1 Deficiency Attenuated the Progression of MesoMT and PF

Because we showed that Tuft1 downregulation blocked induction of MesoMT by diverse mediators, we next evaluated the role of Tuft1 expression in PMCs in our preclinical S. pneumoniae model of PF. To perform this study, we crossed calb2-cre mice with our floxed Tuft1 mice, as reported previously for myocardin (28). The calb2-cre mouse limits cre recombinase expression to calretinin-expressing cells, which in the thoracic compartment is limited to pleural mesothelial cells. This cross-generated a conditional knockout mouse that is Tuft1 deficient in PMCs: Tuft1PMC−/−.

WT and Tuft1PMC−/− were infected with S. pneumoniae via intrapleural injection, as previously reported (3, 4, 21). Injury progressed for 7 days before lung tissues were collected and trichrome stained to determine the extent of pleural injury and fibrosis (Figure 6A). WT mice showed robust pleural thickening after pleural injury. Conversely, pleural thickness was significantly reduced in conditional Tuft1 knockout mice compared with WT treated mice (Figure 6B; P < 0.05). Tissue sections were also stained for MesoMT markers ACTA2 and Col1 (Figures 6C and 6D). Figure 6C and Figure E3 show stitched images of the whole lung stained for ACTA2. The expression of ACTA2 was robust in the pleural mesothelium of S. pneumoniae injured WT mice. Conversely, Tuft1PMC−/− mice demonstrated dramatically less ACTA2 staining throughout the mesothelium. Figure 6D showed intense ACTA2 and Col1 staining in the pleural mesothelium of S. pneumoniae injured WT mice. Conversely, Tuft1PMC−/− mice demonstrated reduced staining of markers of MesoMT. Tuft1 expression was likewise dramatically reduced at the pleural surface when compared with S. pneumoniae injured WT mice (Figure E4). Staining analyses, performed in parallel, showed that calretinin-positive mesothelial cells were at the surface of mouse lung tissues from our preclinical PF model (Figure E5). These studies show that conditional Tuft1 knockout in pleural mesothelial cells protects against the induction of S. pneumoniae–mediated PF in vivo.

Figure 6.

Figure 6.

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Conditional targeting of Tuft1 in the pleural mesothelium reduces the progression of pleural fibrosis. C57BL/6J wild-type and Tuft1PMC−/− mice were intrapleurally administered S. pneumoniae (1.8 × 108) to induce pleural fibrosis. Mice received daily intraperitoneal injections of antibiotics for 4 days. At the end of a 7-day period, lung tissues from injured mice were harvested, fixed in 10% buffered formalin, and processed for histochemical analysis. (A) Lung tissue sections (5 μm) were stained for collagen (blue) and changes in lung morphometry using the Masson’s trichrome staining method. Images were taken at 20× optical zoom. Images presented are representative images of 15 fields per treatment obtained from n = 3–4. (B) Pleural thickness was measured microscopically at random areas covering the entire lung using a Nikon Digital Sight DS-Fi1 camera and NIS Elements BR 3.2 software. *P < 0.05. Lung sections were also (C) stained for ACTA2 (red) and nuclei (blue), and images were stitched to show the entire lung. Images were generated using the Lionheart ETX. Scale bars, 2,000 μm. Images presented are representative images of 15 fields per treatment obtained from n = 3–4. Lung tissue sections were stained for (D) ACTA2 (red), collagen (green), and nuclei (blue). Images were obtained by confocal microscopy (20×). Images presented are representative images of 15 fields per treatment obtained from n = 3–4.

Discussion

Severe pleural injury can lead to the pathological reorganization of the pleural space and extensive PF with symptomatic lung restriction. The mechanisms that drive this process warrant further study. Our prior work and the work of others have shown that pleural mesothelial cells become myofibroblasts in response to treatment with a diverse array of mediators, including TGF-β, thrombin, and FXa (3, 5, 9, 21). Furthermore, the activation of several signaling pathways is critical for the acquisition of this profibrotic phenotype. Although activation of the PI3K/Akt/mTORC1 pathway does not influence MesoMT, the alternate mTOR pathway C2 does (3). Because the mechanism for mTORC2 activation remains unclear, we studied this area comprehensively. Our recently published work carefully detailed the importance of this poorly characterized pathway in the progression of MesoMT and PF (3). Here we show that a newly identified profibrotic protein, Tuft1, contributes to the progression of MesoMT via regulation of mTORC2 signaling in HPMCs. Furthermore, we show that the downregulation of Tuft1 attenuated the progression of PF in a preclinical model of fibrosing pleural disease due to S. pneumoniae infection.

Tuft1 is reported to contribute to the progression of aggressive cellular phenotypes, including diverse forms of cancer (10, 13–17). Furthermore, increased expression of Tuft1 correlated with worse clinical outcomes. However, the contribution of Tuft1 to the progression of PF has not previously been explored. RNA-sequencing analyses identified Tuft1 among other novel genes as being upregulated by TGF-β. Of interest was that our results contradicted publicly available data from single-cell analyses of patients with idiopathic pulmonary fibrosis (IPF; www.ipfcellatalas.com). These databases showed Tuft1 mRNA was unchanged in human fibroblasts and mesothelial cells in IPF versus control lungs. These results again support our assertion that PF is distinctly different from interstitial lung disease. We found that Tuft1 expression is dramatically increased in the pleural mesothelium of pleuritis tissue sections and the pleural surface in our preclinical model compared with normal controls.

Because our prior work showed that diverse mediators induce profibrotic signaling and consequently MesoMT, we next determined the effect of these mediators on Tuft1 expression. As anticipated, mediators of MesoMT induced Tuft1 expression. However, only TGF-β, FXa, and thrombin significantly induced Tuft1. We also found an apparent threshold effect for Tuft1 expression, because >50% knockdown of Tuft1 in the presence of TGF-β was required. Because TGF-β is a potent inducer of Tuft1 (4–10-fold induction), reducing its expression below baseline or control levels proved challenging. Consequently, if Tuft1 expression could not be downregulated below control levels, MesoMT could not be completely blocked (T. Tucker, unpublished results). This observation further underscores the importance of Tuft1 for induction of MesoMT.

Because of the diverse nature of the signaling pathways activated by mediators of MesoMT, the mechanism(s) for Tuft1 induction remain unclear. Work by Kawasaki and colleagues showed that Tuft1 regulated activation of mTORC1 (10, 14). Although mTORC1 is important for fibroblast activation (29), our work has shown that activation of mTORC2, not mTORC1, is critical for MesoMT (3, 10). Although the mechanism of activation for the mTORC2 pathway is unknown, Tuft1 downregulation blunted activation of the mTORC2 pathway. Our prior work has shown that GSK-3β and mTORC2 activation are critical for the induction of MesoMT (3, 8). However, inhibition of GSK-3β blocks the induction of mTORC2 (3). Here we show that GSK-3β inhibition also blocked TGF-β–mediated induction of Tuft1. Conversely, inhibition of mTORC2 had no effect on Tuft1 mRNA expression. These findings, coupled with the ability of enhanced expression of Tuft1 alone to activate mTORC2, suggest that Tuft1 may serve as the switch between GSK-3β and activation of mTORC2. This possibility is under active investigation.

A recent report suggested that Tuft1 expression was enhanced in the bleomycin preclinical model of pulmonary fibrosis (19). However, in this study, enhanced Tuft1 expression was localized to the airway epithelium. In their study, Tuft1 expression was robustly induced by TGF-β in A549 cells. Conversely, TGF-β treatment only modestly induced Tuft1 expression in lung fibroblast cell lines. This study is provocative because the death of airway epithelial cells and the activation and proliferation of fibroblasts are hallmarks of IPF. Although the authors reported that Tuft1 downregulation with adenoviral Tuft1 shRNA could attenuate injury progression, the cell types responsible for this effect were not identified. Our study shows that Tuft1 is a newly identified regulator of MesoMT, which thereby contributes to PF in part by control of the profibrotic HPMC phenotype. Although Niu and colleagues (19) did not fully elucidate this, it is probable that the increased expression of Tuft1 by other cell types, such as fibroblasts, may contribute to the progression of pulmonary disease. These studies are ongoing and fall outside the scope of this work.

Pan-Tuft1 knockout was reported to be lethal, because viable neonates die soon after birth (10). Consequently, we generated floxed Tuft1 mice that were then crossed with calb2-cre–expressing mice. On the basis of our prior work (28), calb2-cre-recombinase targets floxed genes in pleural mesothelial cells, because they are the only cells to express calretinin in the lung compartment. Our work shows that the conditional knockout out of Tuft1 in PMCs protects mice from S. pneumoniae–induced PF.

These novel findings associated with the expression of Tuft1 suggest a potential mechanism for the activation and regulation of the mTORC2 signaling pathway. Specifically, Tuft1 appears to serve as a switch between TGF-β signaling and the activation of the mTORC2 pathway. Our work definitively showed the critical role of mTORC2 in the progression of MesoMT and PF. However, the mechanism of pathway activation remains unclear. However, we report here that mediators such as TGF-β, FXa, and thrombin activate GSK-3β and enhance Tuft1 expression. This expression consequently activates the mTORC2 pathway promoting MesoMT (Figure 7). As such, studies that elucidate the contribution of the likely novel mechanism of action for Tuft1 on mTORC2 activation are warranted and justified by the data presented herein.

Figure 7.

Figure 7.

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Proposed model. Exposure of HPMCs to mediators such as TGF-β, FXa, or thrombin activate GSK-3β via phosphorylation of the tyrosine 216 residue. This activation leads to increased expression of Tuft1. Increased expression of Tuft1 subsequently induces activation of the mTORC2 signaling pathway via phosphorylation of AKT at Ser473. Prepared with BioRender. ECM = extracellular matrix.

Supplemental Materials

Online Data Supplement
rcmb.2024-0263OCS1.docx (3.9MB, docx)
DOI: 10.1165/rcmb.2024-0263OC

Footnotes

Supported by National Institutes of Health grants HL130133 and HL142853 and seed grant funding from the University of Texas Health Science Center at Tyler and The Texas Lung Injury Institute.

Author Contributions: S.O., W.Q., A.J., O.D., S.K., M.F., P.O., L.D., and T.A.T. performed experiments. T.A.T., M.I., and S.I. designed experiments. S.I., M.I., S.K., and T.A.T. prepared the manuscript. All authors fully reviewed and approved the manuscript for submission.

This article has a data supplement, which is accessible at the Supplements tab.

Artificial Intelligence Disclaimer: No artificial intelligence tools were used in writing this manuscript.

Originally Published in Press as DOI: 10.1165/rcmb.2024-0263OC on February 25, 2025

Author disclosures are available with the text of this article at www.atsjournals.org.

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DOI: 10.1165/rcmb.2024-0263OC

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