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Cited by 115TGF-β signaling in diseasesInterstitial Lung Diseases and Idiopathic Pulmonary FibrosisWound Healing and Treatments

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A positive feedback loop bi-stably activates fibroblasts

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Yeo, So-Young, Lee, Keun-Woo, Shin, Dongkwan, An, Sugyun, Cho, Kwang-Hyun, Kim, Seok-Hyung·2018·Nature Communications
DOI 10.1038/s41467-018-05274-6PMC PMC6070563
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Abstract

Although fibroblasts are dormant in normal tissue, they exhibit explosive activation during wound healing and perpetual activation in pathologic fibrosis and cancer stroma. The key regulatory network controlling these fibroblast dynamics is still unknown. Here, we report that Twist1, a key regulator of cancer-associated fibroblasts, directly upregulates Prrx1, which, in turn, increases the expression of Tenascin-C (TNC). TNC also increases Twist1 expression, consequently forming a Twist1-Prrx1-TNC positive feedback loop (PFL). Systems biology studies reveal that the Twist1-Prrx1-TNC PFL can function as a bistable ON/OFF switch and regulates fibroblast activation. This PFL can be irreversibly activated under pathologic conditions, leading to perpetual fibroblast activation. Sustained activation of the Twist1-Prrx1-TNC PFL reproduces fibrotic nodules similar to idiopathic pulmonary fibrosis in vivo and is implicated in fibrotic disease and cancer stroma. Considering that this PFL is specific to activated fibroblasts, Twist1-Prrx1-TNC PFL may be a fibroblast-specific therapeutic target to deprogram perpetually activated fibroblasts.

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Fig. 1 Twist1 increases tenascin-c expression in cancer-associated fibroblasts. Twist1 is a potent but indirect inducer of tenascin-c (TNC), which is essential for maintaining Twist1 expression in cancer-associated fibroblasts (CAFs). a Normal fibroblasts were transduced with Twist1 encoding lentivirus or GFP control. Induced Twist1 expression increased both the protein and mRNA of TNC in stomach normal fibroblasts (SNF#14 and SNF#32) and esophageal normal fibroblasts (ENF#8). CAFs were transduced with lentivirus-expressing shRNA specific for Twist1 (shTw1) or nonspecific shRNA (shNS). Knockdown of Twist1 also decreased the TNC expression in the esophageal and stomach cancer-derived CAFs (ECAF#8, SCAF#14, and SCAF#32). Real-time RT-PCR data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: **p<0.001, ***p<0.0001). b We transfected HT1080 cells with TNC promoter-driven (−938~ +112bp) luciferase reporter genes simultaneously with a Twist1 expression vector. Although we added Twist1, the luciferase reporter assay revealed that Twist1 did not enhance the TNC promoter activity. Data are presented as the mean ± SEM; N = 4 independent experiments (two-tailed t test: ns not significant). c The chromatin immunoprecipitation assay indicated that Twist1 did not bind to any E-box sequences between −3.8kb and +25bp of the TNC transcription start sites. In contrast, Twist1 bound to independent positive controls such as E-box sequences in the PDGFRa and SDF1a promoter (ChIP P.C-PDGFRa / SDF1a) but not to negative control, non E-box sequence in SDF1a promoter (ChIP N.C.-SDF1a). Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). d The shRNA-mediated depletion of TNC led to decreased colony formation and gel contraction abilities in stomach cancer-derived CAFs (SCAF#36). The TNC silencing also increased the proportion of apoptotic cells. e Exogenous TNC expression in normal fibroblasts (SNF#19) enhanced the Twist1 expression, whereas the TNC depletion in stomach cancer-derived CAFs (SCAF#14 and #32) led to Twist1 downregulation. f TNC-induced Twist1 expression was mediated by Integrin β3. LM609 (Integrin β3 blocking Ab) abrogated TNC-mediated Twist1 expression in normal fibroblasts (SNF#19). g Schematic diagram of the feedback regulation between Twist1 and TNC
Fig. 1 Twist1 increases tenascin-c expression in cancer-associated fibroblasts. Twist1 is a potent but indirect inducer of tenascin-c (TNC), which is essential for maintaining Twist1 expression in cancer-associated fibroblasts (CAFs). a Normal fibroblasts were transduced with Twist1 encoding lentivirus or GFP control. Induced Twist1 expression increased both the protein and mRNA of TNC in stomach normal fibroblasts (SNF#14 and SNF#32) and esophageal normal fibroblasts (ENF#8). CAFs were transduced with lentivirus-expressing shRNA specific for Twist1 (shTw1) or nonspecific shRNA (shNS). Knockdown of Twist1 also decreased the TNC expression in the esophageal and stomach cancer-derived CAFs (ECAF#8, SCAF#14, and SCAF#32). Real-time RT-PCR data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: **p<0.001, ***p<0.0001). b We transfected HT1080 cells with TNC promoter-driven (−938~ +112bp) luciferase reporter genes simultaneously with a Twist1 expression vector. Although we added Twist1, the luciferase reporter assay revealed that Twist1 did not enhance the TNC promoter activity. Data are presented as the mean ± SEM; N = 4 independent experiments (two-tailed t test: ns not significant). c The chromatin immunoprecipitation assay indicated that Twist1 did not bind to any E-box sequences between −3.8kb and +25bp of the TNC transcription start sites. In contrast, Twist1 bound to independent positive controls such as E-box sequences in the PDGFRa and SDF1a promoter (ChIP P.C-PDGFRa / SDF1a) but not to negative control, non E-box sequence in SDF1a promoter (ChIP N.C.-SDF1a). Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). d The shRNA-mediated depletion of TNC led to decreased colony formation and gel contraction abilities in stomach cancer-derived CAFs (SCAF#36). The TNC silencing also increased the proportion of apoptotic cells. e Exogenous TNC expression in normal fibroblasts (SNF#19) enhanced the Twist1 expression, whereas the TNC depletion in stomach cancer-derived CAFs (SCAF#14 and #32) led to Twist1 downregulation. f TNC-induced Twist1 expression was mediated by Integrin β3. LM609 (Integrin β3 blocking Ab) abrogated TNC-mediated Twist1 expression in normal fibroblasts (SNF#19). g Schematic diagram of the feedback regulation between Twist1 and TNCCC BY · PubMed Central
Fig. 2 Twist1 was an essential, positive transcriptional regulator of Prrx1 expression in both normal fibroblasts and CAFs. Twist1 enhanced the expression of Prrx1, a potential TNC inducer, by direct transcriptional activation. a Analysis of the FANTOM database revealed 11 candidate genes whose expressions were significantly correlated with both Twist1 and TNC expression in fibroblasts and stem cells. b Induction of Twist1 expression increased Prrx1 expression in normal fibroblasts. c The knockdown of Twist1 expression in CAFs led to decreased Prrx1 expression. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: **p<0.001, ***p<0.0001). d To determine whether Twist1 regulates Prrx1 promoter activity, Prrx1 promoter-driven luciferase gene and Twist1 expression vector were cotransfected into HT1080 fibrosarcoma cells. The luciferase reporter assay revealed that the ectopic expression of Twist1 increased the Prrx1 promoter activity. Of the four different Prrx1 promoter constructs, construct #2 containing two E-boxes between −798 and −592 bp from the TSS of Prrx1 showed the most obvious difference in response to transient cotransfection with a Twist1 expression vector. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.001). e Two E-box mutations in E-box 1 (CATCTG (−619) to GGTCTG) and E-box 2 (CAACTG (−787) to TCACTG) were introduced in HT1080 cells. The Twist1-induced Prrx1 promoter activity was negated by the E-box mutation that was located at 619 bp of the TSS of Prrx1. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). f The schematic illustration of the ChIP assay PCR primer binding sites in the Prrx1 promoter region. We used the E-box sequence in the SDF1a promoter as a positive control. Compared with the IgG and positive controls, the Prrx1 promoter region that contained E-box 1 and E-box 2 was specifically enriched by each primer set after chromatin immunoprecipitation. That is, the ChIP assay revealed that Twist1 binds to the Prrx1 promoter sites (−819 to −597 bp) that contain two E-boxes. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). g Schematic diagram of the regulatory relationship among Twist1, Prrx1, and TNC
Fig. 2 Twist1 was an essential, positive transcriptional regulator of Prrx1 expression in both normal fibroblasts and CAFs. Twist1 enhanced the expression of Prrx1, a potential TNC inducer, by direct transcriptional activation. a Analysis of the FANTOM database revealed 11 candidate genes whose expressions were significantly correlated with both Twist1 and TNC expression in fibroblasts and stem cells. b Induction of Twist1 expression increased Prrx1 expression in normal fibroblasts. c The knockdown of Twist1 expression in CAFs led to decreased Prrx1 expression. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: **p<0.001, ***p<0.0001). d To determine whether Twist1 regulates Prrx1 promoter activity, Prrx1 promoter-driven luciferase gene and Twist1 expression vector were cotransfected into HT1080 fibrosarcoma cells. The luciferase reporter assay revealed that the ectopic expression of Twist1 increased the Prrx1 promoter activity. Of the four different Prrx1 promoter constructs, construct #2 containing two E-boxes between −798 and −592 bp from the TSS of Prrx1 showed the most obvious difference in response to transient cotransfection with a Twist1 expression vector. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.001). e Two E-box mutations in E-box 1 (CATCTG (−619) to GGTCTG) and E-box 2 (CAACTG (−787) to TCACTG) were introduced in HT1080 cells. The Twist1-induced Prrx1 promoter activity was negated by the E-box mutation that was located at 619 bp of the TSS of Prrx1. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). f The schematic illustration of the ChIP assay PCR primer binding sites in the Prrx1 promoter region. We used the E-box sequence in the SDF1a promoter as a positive control. Compared with the IgG and positive controls, the Prrx1 promoter region that contained E-box 1 and E-box 2 was specifically enriched by each primer set after chromatin immunoprecipitation. That is, the ChIP assay revealed that Twist1 binds to the Prrx1 promoter sites (−819 to −597 bp) that contain two E-boxes. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.05). g Schematic diagram of the regulatory relationship among Twist1, Prrx1, and TNCCC BY · PubMed Central
Fig. 3 Twist1, Prrx1, and TNC formed a positive feedback loop in fibroblasts. a, b Prrx1 directly upregulates TNC expression in fibroblasts. a Both Prrx1a and Prrx1b increased the TNC protein level in normal fibroblasts (SNF#14 and #32). Prrx1 depletion led to the downregulation of TNC expression in CAFs (SCAF#32 and #39). b Prrx1 enhanced the TNC promoter activity. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.01). c, d Prrx1 is indispensable for Twist1-induced TNC expression at the protein (c) and mRNA (d) level. Data are presented as the mean ± SEM; N = 4 independent experiments (two-tailed t test: ***p<0.0001). e, f To confirm the Twist1-Prrx1-TNC PFL, we introduced a doxycycline-inducible Twist1 expression vector into two fibroblast cell lines (MRC5 and IMR90) as well as a normal fibroblast line (SNF#19). Induced Twist1 expression using this Tet-ON system resulted in sequential increases of the Prrx1 and TNC protein (e) and mRNA (f) expression in normal lung fibroblasts (MRC5). g Schematic diagram of the Twist1-Prrx1-TNC PFL
Fig. 3 Twist1, Prrx1, and TNC formed a positive feedback loop in fibroblasts. a, b Prrx1 directly upregulates TNC expression in fibroblasts. a Both Prrx1a and Prrx1b increased the TNC protein level in normal fibroblasts (SNF#14 and #32). Prrx1 depletion led to the downregulation of TNC expression in CAFs (SCAF#32 and #39). b Prrx1 enhanced the TNC promoter activity. Data are presented as the mean ± SEM; N = 3 independent experiments (two-tailed t test: *p<0.01). c, d Prrx1 is indispensable for Twist1-induced TNC expression at the protein (c) and mRNA (d) level. Data are presented as the mean ± SEM; N = 4 independent experiments (two-tailed t test: ***p<0.0001). e, f To confirm the Twist1-Prrx1-TNC PFL, we introduced a doxycycline-inducible Twist1 expression vector into two fibroblast cell lines (MRC5 and IMR90) as well as a normal fibroblast line (SNF#19). Induced Twist1 expression using this Tet-ON system resulted in sequential increases of the Prrx1 and TNC protein (e) and mRNA (f) expression in normal lung fibroblasts (MRC5). g Schematic diagram of the Twist1-Prrx1-TNC PFLCC BY · PubMed Central
Fig. 4 The Twist1-Prrx1-TNC PFL functions as an internal bistable switch in regulating the proliferative signal of fibroblasts. The importance and functional implications of the Twist1-Prrx1-TNC PFL were demonstrated through mathematical simulations based on experimental data. a Schematic diagram of the Twist1-Prrx1-TNC PFL and the mathematical description of the loop (see Supplementary text for detailed explanations). b Ultrasensitive responses of the PFL to the varying doxycycline stimuli compared with cases in which feedback regulation was absent (VTNC→Tw = 0). c Hysteretic responses of the three genes to the doxycycline stimulus. d Various profiles for the hysteretic TNC response to the doxycycline stimulation along with the increase of the feedback strength (VTNC→Tw). e Irreversible hysteretic responses of the three genes to the doxycycline stimulus when the feedback strength is large (VTNC→Tw = 80). f Phase diagram illustrating the irreversibility of TNC with respect to the feedback strength (VTNC→Tw) and stability of Twist1 (KTw⁺∕KTw⁻). ΔTNC denotes TNCfin − TNCini when doxycycline = 0 (left). Reversibility was characterized by TNC = 0 (irreversible, otherwise). The irreversibility of TNC can be categorized into two phases (center). Representative hysteretic curves for the corresponding phases are shown in the right panel
Fig. 4 The Twist1-Prrx1-TNC PFL functions as an internal bistable switch in regulating the proliferative signal of fibroblasts. The importance and functional implications of the Twist1-Prrx1-TNC PFL were demonstrated through mathematical simulations based on experimental data. a Schematic diagram of the Twist1-Prrx1-TNC PFL and the mathematical description of the loop (see Supplementary text for detailed explanations). b Ultrasensitive responses of the PFL to the varying doxycycline stimuli compared with cases in which feedback regulation was absent (VTNC→Tw = 0). c Hysteretic responses of the three genes to the doxycycline stimulus. d Various profiles for the hysteretic TNC response to the doxycycline stimulation along with the increase of the feedback strength (VTNC→Tw). e Irreversible hysteretic responses of the three genes to the doxycycline stimulus when the feedback strength is large (VTNC→Tw = 80). f Phase diagram illustrating the irreversibility of TNC with respect to the feedback strength (VTNC→Tw) and stability of Twist1 (KTw⁺∕KTw⁻). ΔTNC denotes TNCfin − TNCini when doxycycline = 0 (left). Reversibility was characterized by TNC = 0 (irreversible, otherwise). The irreversibility of TNC can be categorized into two phases (center). Representative hysteretic curves for the corresponding phases are shown in the right panelCC BY · PubMed Central
Fig. 5 The role of the Twist1-Prrx1-TNC PFL as a bistable switch was validated using an in vitro fibroblast line, cutaneous wound healing, and patient-derived CAFs. a Twist1 expression was induced in fibroblasts (MRC5) using a doxycycline-mediated induction system, and we examined the impact of the induced Twist1 on the Prrx1 and TNC expression profiles using multiplex immunohistochemistry (mIHC). Bimodal distribution of the triple gene expression, such as all positive vs. all negative, was evident after 7 days of induction. The insert images are enlarged picture of stained cells in each staining images. b We examined the Twist1, Prrx1, and TNC expressions during cutaneous wound healing using multiplex immunohistochemistry. The yellow box is a low power view of the mouse wound area. In this yellow box, dashed circle and arrow indicate area of interest (AOI) which is shown as a high power view. c Quantitative analysis of Twist1, Prrx1, and TNC expression in fibroblasts during wound healing. After we made circle-full-thickness cutaneous wounds (diameter of 5 mm) on the dorsal skin of mice, we determined the Twist1, Prrx1, and TNC expression in fibroblasts at multiple time points using mIHC. We noted highly dynamic changes in these three genes’ expression in fibroblasts. d The plot is generated based on data derived from Fig. 5b. Fibroblasts that were positive for all three genes were restricted to the granulation tissue formation stage of wound healing. e The GFP reporter, under the control of a Twist1 promoter, was stably integrated into the chromosome of two patient-derived CAFs. Then, we sorted CAFs into Twist1-positive and Twist1-negative CAFs, using flow cytometry. f Esophageal cancer-derived CAFs (ECAF#8) were sorted into Twist1-positive and Twist1-negative CAFs. Twist1-positive CAFs were specifically positive for Prrx1 and TNC and also exhibited increased colony-forming abilities. g The Twist1-high CAF (ECAF#8) showed resistance to doxorubicin-induced apoptosis
Fig. 5 The role of the Twist1-Prrx1-TNC PFL as a bistable switch was validated using an in vitro fibroblast line, cutaneous wound healing, and patient-derived CAFs. a Twist1 expression was induced in fibroblasts (MRC5) using a doxycycline-mediated induction system, and we examined the impact of the induced Twist1 on the Prrx1 and TNC expression profiles using multiplex immunohistochemistry (mIHC). Bimodal distribution of the triple gene expression, such as all positive vs. all negative, was evident after 7 days of induction. The insert images are enlarged picture of stained cells in each staining images. b We examined the Twist1, Prrx1, and TNC expressions during cutaneous wound healing using multiplex immunohistochemistry. The yellow box is a low power view of the mouse wound area. In this yellow box, dashed circle and arrow indicate area of interest (AOI) which is shown as a high power view. c Quantitative analysis of Twist1, Prrx1, and TNC expression in fibroblasts during wound healing. After we made circle-full-thickness cutaneous wounds (diameter of 5 mm) on the dorsal skin of mice, we determined the Twist1, Prrx1, and TNC expression in fibroblasts at multiple time points using mIHC. We noted highly dynamic changes in these three genes’ expression in fibroblasts. d The plot is generated based on data derived from Fig. 5b. Fibroblasts that were positive for all three genes were restricted to the granulation tissue formation stage of wound healing. e The GFP reporter, under the control of a Twist1 promoter, was stably integrated into the chromosome of two patient-derived CAFs. Then, we sorted CAFs into Twist1-positive and Twist1-negative CAFs, using flow cytometry. f Esophageal cancer-derived CAFs (ECAF#8) were sorted into Twist1-positive and Twist1-negative CAFs. Twist1-positive CAFs were specifically positive for Prrx1 and TNC and also exhibited increased colony-forming abilities. g The Twist1-high CAF (ECAF#8) showed resistance to doxorubicin-induced apoptosisCC BY · PubMed Central
Fig. 6 Sustained switching ON of the Twist1-Prrx1-TNC PFL triggers persistent fibroblast activation, forming fibrotic nodules in vivo during wound healing. a Schematic diagram of the experimental design. We injected TNC protein or PBS (control) into the wound area of WT and fibroblast-specific Twist1 knockout mice. The experiments were conducted three times (N = 5 mice in each experimental group). Experimental timeline for the detailed procedures is indicated. b The maintenance of the PFL activation by TNC injection (FSP1-cre; Twist1 WT mice, left) and disruption of PFL by fibroblast-specific Twist1 deficiency despite the TNC injection (FSP1- Cre;Twist1fl/fl mice, right). c H&E staining images showing the wound areas of each group (top). In Twist1 WT mice (depicted as iii, FSP1-cre; Twist1 WT), we observed fibroblastic nodules (red arrow) comprising hyper-proliferative immature fibroblasts and epithelial cell hyperplasia (blue dotted line). TNC is strongly expressed in most fibroblastic nodules as mentioned above in which Twist1 and Prrx1 are also strongly coexpressed. These results were obtained through multiplex IHC staining and visualized using ImageJ Fiji (Twist1-green, Prrx1-red, TNC-yellow, and hematoxylin-blue). We performed quantitative analysis of these results using FCS Express 6 image cytometry analysis software, and we presented the outcomes as an FACS plot (x axis-Twist1 green, y axis-Prrx1 red and contour indicated with TNC) (bottom). The number of fibroblasts displaying Twist1/Prrx1 double positivity in the TNC- or PBS-injected wound areas (150 μm × 150 μm square) is indicated by box plot (bottom right of Fig. 6c). Data are presented as the mean ± SEM; N = 6 independent measurements (two-tailed t test: **p<0.005, ***p<0.001)
Fig. 6 Sustained switching ON of the Twist1-Prrx1-TNC PFL triggers persistent fibroblast activation, forming fibrotic nodules in vivo during wound healing. a Schematic diagram of the experimental design. We injected TNC protein or PBS (control) into the wound area of WT and fibroblast-specific Twist1 knockout mice. The experiments were conducted three times (N = 5 mice in each experimental group). Experimental timeline for the detailed procedures is indicated. b The maintenance of the PFL activation by TNC injection (FSP1-cre; Twist1 WT mice, left) and disruption of PFL by fibroblast-specific Twist1 deficiency despite the TNC injection (FSP1- Cre;Twist1fl/fl mice, right). c H&E staining images showing the wound areas of each group (top). In Twist1 WT mice (depicted as iii, FSP1-cre; Twist1 WT), we observed fibroblastic nodules (red arrow) comprising hyper-proliferative immature fibroblasts and epithelial cell hyperplasia (blue dotted line). TNC is strongly expressed in most fibroblastic nodules as mentioned above in which Twist1 and Prrx1 are also strongly coexpressed. These results were obtained through multiplex IHC staining and visualized using ImageJ Fiji (Twist1-green, Prrx1-red, TNC-yellow, and hematoxylin-blue). We performed quantitative analysis of these results using FCS Express 6 image cytometry analysis software, and we presented the outcomes as an FACS plot (x axis-Twist1 green, y axis-Prrx1 red and contour indicated with TNC) (bottom). The number of fibroblasts displaying Twist1/Prrx1 double positivity in the TNC- or PBS-injected wound areas (150 μm × 150 μm square) is indicated by box plot (bottom right of Fig. 6c). Data are presented as the mean ± SEM; N = 6 independent measurements (two-tailed t test: **p<0.005, ***p<0.001)CC BY · PubMed Central
Fig. 7 High TNC is essential for activating the irreversible PFL. a In the fibrotic nodule generated by exogenous TNC, we observed a TNC concentration gradient (top). In-depth analysis of this nodule using multiplex IHC showed that Twist1 and Prrx1 were strongly positive and coexpressed in most fibroblasts within strongly TNC-positive zone1, and this pattern disappeared as the TNC concentration decreased (bottom left) (Twist1-green, Prrx1-red, TNC-yellow and hematoxylin-blue). We measured each integrated density per zone representing the expression level of each gene using ImageJ Fiji (bottom right). Data are presented as the mean ± SEM; N = 3 independent measurements (two-tailed t test: *p<0.05, **p<0.001, ***p<0.0001). b We treated mouse dermal fibroblasts isolated from fresh Balb/c mouse tissue with TNC at different concentrations (0, 0.1, 1, 2, and 5 μg) and then evaluated Twist1 and TNC expression by western blotting (top). We estimated the kinetic parameter values of the mathematical model based on this western blotting data on the Twist1 and TNC fold changes along with the increase of exogenous TNC (bottom). Discrete data marked with squares indicate experimental measurements of the corresponding genes (N = 3, error bars indicate s.d.). c Phase diagram illustrating the irreversibility of TNC (represented by color intensity) with respect to the feedback strength (V) and the TNC level for feedback activation (Km1, the Michaelis−Menten constant) (top left). The hysteretic responses of TNC to the TNC injection in the irreversible ((Km1, V) = (5, 30)) and reversible ((Km1, V) = (10, 15)) states are shown in the top right and bottom left figures, respectively. Given that the stimulus increases from zero to TNCmax and then decreases back to zero, we compared the irreversibility of TNC, ∆TNC, with that of the reversible state (bottom right). The inlet shows examples of obtaining ∆TNC for the TNCmax
Fig. 7 High TNC is essential for activating the irreversible PFL. a In the fibrotic nodule generated by exogenous TNC, we observed a TNC concentration gradient (top). In-depth analysis of this nodule using multiplex IHC showed that Twist1 and Prrx1 were strongly positive and coexpressed in most fibroblasts within strongly TNC-positive zone1, and this pattern disappeared as the TNC concentration decreased (bottom left) (Twist1-green, Prrx1-red, TNC-yellow and hematoxylin-blue). We measured each integrated density per zone representing the expression level of each gene using ImageJ Fiji (bottom right). Data are presented as the mean ± SEM; N = 3 independent measurements (two-tailed t test: *p<0.05, **p<0.001, ***p<0.0001). b We treated mouse dermal fibroblasts isolated from fresh Balb/c mouse tissue with TNC at different concentrations (0, 0.1, 1, 2, and 5 μg) and then evaluated Twist1 and TNC expression by western blotting (top). We estimated the kinetic parameter values of the mathematical model based on this western blotting data on the Twist1 and TNC fold changes along with the increase of exogenous TNC (bottom). Discrete data marked with squares indicate experimental measurements of the corresponding genes (N = 3, error bars indicate s.d.). c Phase diagram illustrating the irreversibility of TNC (represented by color intensity) with respect to the feedback strength (V) and the TNC level for feedback activation (Km1, the Michaelis−Menten constant) (top left). The hysteretic responses of TNC to the TNC injection in the irreversible ((Km1, V) = (5, 30)) and reversible ((Km1, V) = (10, 15)) states are shown in the top right and bottom left figures, respectively. Given that the stimulus increases from zero to TNCmax and then decreases back to zero, we compared the irreversibility of TNC, ∆TNC, with that of the reversible state (bottom right). The inlet shows examples of obtaining ∆TNC for the TNCmaxCC BY · PubMed Central
Fig. 8 The coexpression of Twist1, Prrx1, and TNC is frequently found in cancer-associated fibroblasts and pathologic fibrosis. The clinical implications of the Twist1-Prrx1-TNC PFL in CAFs and fibrotic diseases. a, b We examined the coexpressions of all three genes by mIHC (scale bar indicates 100 μm). The bimodal distribution of the triple gene expression, all positive vs. all negative, a hallmark of a bistable system, was evident again in esophageal CAFs. These data are highly consistent with the computational prediction that the Twist1-Prrx1-TNC PFL functions may lead to irreversible and perpetual activation in pathologic conditions. We analyzed the expression profiles of these three genes in 306 esophageal cancer tissues. c The presence of CAFs that were positive for all three genes was strongly associated with the worse clinical outcomes. d Fibroblasts that were positive for all three genes were frequently observed in pathologic fibrosis, such as idiopathic pulmonary fibrosis, keloid disease, and desmoid tumors (aggressive fibromatosis). Scale bar indicates 100 μm
Fig. 8 The coexpression of Twist1, Prrx1, and TNC is frequently found in cancer-associated fibroblasts and pathologic fibrosis. The clinical implications of the Twist1-Prrx1-TNC PFL in CAFs and fibrotic diseases. a, b We examined the coexpressions of all three genes by mIHC (scale bar indicates 100 μm). The bimodal distribution of the triple gene expression, all positive vs. all negative, a hallmark of a bistable system, was evident again in esophageal CAFs. These data are highly consistent with the computational prediction that the Twist1-Prrx1-TNC PFL functions may lead to irreversible and perpetual activation in pathologic conditions. We analyzed the expression profiles of these three genes in 306 esophageal cancer tissues. c The presence of CAFs that were positive for all three genes was strongly associated with the worse clinical outcomes. d Fibroblasts that were positive for all three genes were frequently observed in pathologic fibrosis, such as idiopathic pulmonary fibrosis, keloid disease, and desmoid tumors (aggressive fibromatosis). Scale bar indicates 100 μmCC BY · PubMed Central
Fig. 9 Summary of our study. The Twist1-Prrx1-TNC positive feedback regulation provides clues for understanding the activation of fibroblasts during wound healing under normal conditions, as well as abnormally activated fibroblasts in pathological conditions such as cancerous and fibrotic diseases. Under normal conditions, the PFL acts as a reversible bistable switch by which the activation of fibroblasts is “ON" above a sufficient level of stimulation and “OFF" for the withdrawal of the stimulus. However, this switch can be permanently turned on under pathologic conditions by continued activation of the PFL, resulting in sustained proliferation of fibroblasts
Fig. 9 Summary of our study. The Twist1-Prrx1-TNC positive feedback regulation provides clues for understanding the activation of fibroblasts during wound healing under normal conditions, as well as abnormally activated fibroblasts in pathological conditions such as cancerous and fibrotic diseases. Under normal conditions, the PFL acts as a reversible bistable switch by which the activation of fibroblasts is “ON" above a sufficient level of stimulation and “OFF" for the withdrawal of the stimulus. However, this switch can be permanently turned on under pathologic conditions by continued activation of the PFL, resulting in sustained proliferation of fibroblastsCC BY · PubMed Central

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Authors
Yeo, S.-Y., Lee, K.-W., Shin, D., An, S., Cho, K.-H., & Kim, S.-H.
Affiliations
  • Sungkyunkwan University (KR)
  • Korea Advanced Institute of Science and Technology (KR)
  • Samsung Medical Center (KR)
Corresponding author
  • Cho, Kwang-Hyun
  • Kim, Seok-Hyung
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-08-01
Volume
9
Issue
1
Pages
3016-3016
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-05274-6
PMC
PMC6070563
OpenAlex
W2883191015
Cited by
115
References
57
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4.00
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journal article
Language
en
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  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Research Foundation (2015R1A2A1A15054021)
  • Ministry of Health, Welfare and Family Affairs | Korea National Institute of Health (HI14C2517)
  • National Research Foundation of Korea (2017R1A2A1A17069642, 2015M3A9A7067220, 2013M3A9A7046303)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
  • Health Sciences › Medicine › Surgery › Wound Healing and Treatments
Keywords
fibroblast activation, positive feedback loop, TWIST1, PRRX1, tenascin-C, bistability, cancer-associated fibroblasts, fibrosis, idiopathic pulmonary fibrosis, tumor stroma, wound healing, gene regulatory networks
MeSH terms
Cancer-Associated Fibroblasts, Animals, Cell Line, Feedback, Fibroblasts, Fibrosis, Humans, Mice, Inbred BALB C, Nuclear Proteins, Homeodomain Proteins, Tenascin, Systems Biology, Twist-Related Protein 1

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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