refined-murine-model-of-idiopathic-pulmonary-fibrosis

Refined Murine Model of Idiopathic Pulmonary Fibrosis

Animal models play a crucial role in understanding disease mechanisms and testing potential therapeutic approaches. However, traditional murine models of ...

Sun Yat-sen University

Lipotoxicity leads to insulin secretion deficiency, which is among the important causes for the onset of type 2 diabetes mellitus. Thus, the restoration of β-cell mass and preservation of its endocrine function are long-sought goals in diabetes research. Previous studies have suggested that the membrane protein caveolin-1 (Cav-1) is implicated in β-cell apoptosis and insulin secretion, however, the underlying mechanisms still remains unclear. Our objective is to explore whether Cav-1 depletion protects pancreatic β cells from lipotoxicity and what are the underlying mechanisms. In this study, we found that Cav-1 silencing significantly promoted β-cell proliferation, inhibited palmitate (PA)-induced pancreatic β-cell apoptosis and enhanced insulin production and secretion. These effects were associated with enhanced activities of Akt and ERK1/2, which in turn downregulated the expression of cell cycle inhibitors (FOXO1, GSK3β, P21, P27 and P53) and upregulated the expression of Cyclin D2 and Cyclin D3. Subsequent inhibition of PI3K/Akt and ERK/MAPK pathways abolished Cav-1 depletion induced β-cell mass protection. Furthermore, under PA induced endoplasmic reticulum (ER) stress, Cav-1 silencing significantly reduced eIF2α phosphorylation and the expression of ER stress-responsive markers BiP and CHOP, which are among the known sensitizers of lipotoxicity. Our findings suggest Cav-1 as potential target molecule in T2DM treatment via the preservation of lipotoxicity-induced β-cell mass reduction and the attenuation of insulin secretion dysfunction.

Caveolin-1 deficiency protects pancreatic β cells against palmitate-induced dysfunction and apoptosis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), and is highly contagious and pathogenic. TMPRSS2 and Neuropilin-1, the key components that facilitate SARS-CoV-2 infection, are potential targets for treatment of COVID-19. Here we performed a comprehensive analysis on NRP1 and TMPRSS2 in lung to provide information for treating comorbidity of COVID-19 with lung cancer. NRP1 is widely expressed across all the human tissues while TMPRSS2 is expressed in a restricted pattern. High level of NRP1 associates with worse prognosis in multiple cancers, while high level of TMPRSS2 is associated with better survival of Lung Adenocarcinoma (LUAD). Moreover, NRP1 positively correlates with the oncogenic Cancer Associated Fibroblast (CAF), macrophage and endothelial cells infiltration, negatively correlates with infiltration of CD8 T cell, the tumor killer cell in Lung Squamous cell carcinoma (LUSC). TMPRSS2 shows negative correlation with the oncogenic events in LUAD. RNA-seq data show that NRP1 level is slightly decreased in peripheral blood of ICU admitted COVID-19 patients, unaltered in lung, while TMPRSS2 level is significantly decreased in lung of COVID-19 patients. Our analysis suggests NRP1 as a potential therapeutic target, while sets an alert on targeting TMPRSS2 for treating comorbidity of COVID-19 and lung cancers.

 analysis identifies neuropilin-1 as a potential therapeutic target for SARS-Cov-2 infected lung cancer patients.

Insulin treatment was confirmed to reduce insulin resistance, but the underlying mechanism remains unknown. Caveolin-1 (Cav-1) is a functional protein of the membrane lipid rafts, known as caveolae, and is widely expressed in mammalian adipose tissue. There is increasing evidence that show the involvement of Cav-1 in the AKT activation, which is responsible for insulin sensitivity. Our aim was to investigate the effect of Cav-1 depletion on insulin sensitivity and AKT activation in glargine-treated type 2 diabetic mice. Mice were exposed to a high-fat diet and subject to intraperitoneal injection of streptozotocin to induce diabetes. Next, glargine was administered to treat T2DM mice for 3 weeks (insulin group). The expression of Cav-1 was then silenced by injecting lentiviral-vectored short hairpin RNA (shRNA) through the tail vein of glargine-treated T2DM mice (CAV1-shRNA group), while scramble virus injection was used as a negative control (Ctrl-shRNA group). The results showed that glargine was able to upregulate the expression of PI3K and activate serine phosphorylation of AKT through the upregulation of Cav-1 expression in paraepididymal adipose tissue of the insulin group. However, glargine treatment could not activate AKT pathway in Cav-1 silenced diabetic mice. These results suggest that Cav-1 is essential for the activation of AKT and improving insulin sensitivity in type 2 diabetic mice during glargine treatment.

Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Lung squamous cell carcinoma (LUSC) causes over 400,000 deaths annually, yet it lacks targeted therapy. A major antagonist of Hedgehog pathway, HHIP (Hedgehog Interacting Protein) plays an important role in LUSC; however, the regulatory mechanism remains unclear. Long non-coding RNA HHIP-AS1 plays suppressive or promotive roles in different cancers, but its role in LUSC remains unknown. This manuscript is to investigate regulatory mechanism of HHIP and the role of HHIP-AS1 in LUSC.

LncRNA HHIP-AS1 suppresses lung squamous cell carcinoma by stabilizing HHIP mRNA.

The typical function of Drosha is participating in cleaving pri-miRNA, the initial step of miRNA biogenesis, in the nucleus. Since Drosha has a double-stranded RNA-binding domain and two RNase III domains, when it binds and/or cleaves other RNA species other than pri-miRNA, Drosha is able to induce a variety of novel biological effects. Moreover, by interacting with other protein, Drosha is able to modify the function of other protein complexes. Recently, diverse non-classical functions of Drosha have been demonstrated, such as promoting DNA damage repair, transcriptional activation and inhibition, pre-mRNA splicing regulation, mRNA destabilization, and virus-host interaction. In this review, we describe these newly discovered functions of Drosha in order to present a panoramic picture of the novel biological processes that Drosha is involved in.

Review: Non-canonical role of Drosha ribonuclease III.

Lung squamous cell carcinoma (LUSC) accounts for approximately 25% to 30% of lung cancers, but largely no targeted therapy is available against it, calling for identification of new oncogenes in LUSC growth for new therapeutic targets. In this study, REL was identified through a screening for oncogenes that are highly amplified in human LUSC. Its expression was associated with poor prognosis in LUSC patients. Furthermore, knockdown of c-Rel in LUSC cell lines lead to significant decrease in cell proliferation and migration. Mechanistically, c-Rel knockdown suppressed NFκB pathway by blocking phosphorylation of IκB. Consistently, pharmaceutic inhibition of c-Rel also. In orthotopic xenograft lung cancer mouse model, c-Rel knockdown inhibited the tumor growth. Cancer cell proliferation and epithelial-mesenchymal-transition (EMT) of the tumors were impaired by c-Rel knockdown. Finally, it's confirmed in precision-cut tumor slices of LUSC that deletion of c-Rel inhibits the NFκB pathway and cancer cell growth. Accordingly, we hypothesize that c-Rel promotes the activation of the NFκB pathway by promoting the phosphorylation of IκB in LUSC. Our study reveals REL as a novel LUSC oncogene and provides new insights into the molecular regulation of LUSC, which will provide new therapeutic targets for the treatment of squamous lung cancer.

c-Rel is a Novel Oncogene in Lung Squamous Cell Carcinoma Regulating Cell Proliferation and Migration.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no cure except transplantation. Abnormal alveolar epithelial regeneration is a key driver of IPF development. The function of Yes1 Associated Transcriptional Regulator (YAP) in alveolar regeneration and IPF pathogenesis remains elusive. Here, we first revealed the activation of YAP in alveolar epithelium 2 cells (AEC2s) from human IPF lungs and fibrotic mouse lungs. Notably, conditional deletion of YAP in mouse AEC2s exacerbated bleomycin-induced pulmonary fibrosis. Intriguingly, we showed in both conditional knockout mice and alveolar organoids that YAP deficiency impaired AEC2 proliferation and differentiation into alveolar epithelium 1 cells (AEC1s). Mechanistically, YAP regulated expression levels of genes associated with cell cycle progression and AEC1 differentiation. Furthermore, overexpression of YAP in vitro promoted cell proliferation. These results indicate the critical role of YAP in alveolar regeneration and IPF pathogenesis. Our findings provide new insights into the regulation of alveolar regeneration and IPF pathogenesis, paving the road for developing novel treatment strategies.

YAP Alleviates Pulmonary Fibrosis Through Promoting Alveolar Regeneration via Modulating the Stemness of Alveolar Type 2 Cells.

Airway mucous cell metaplasia is a significant feature of many chronic airway diseases, such as chronic obstructive pulmonary disease, cystic fibrosis and asthma. However, the mechanisms underlying this process remain poorly understood. Here, we employed in vivo mouse genetic models to demonstrate that Hippo and p53 (encoded by Trp53) cooperate to modulate the differentiation of club cells into goblet cells. We revealed that ablation of Mst1 (Stk4) and Mst2 (Stk3), encoding the core components of Hippo signaling, significantly reduces mucous metaplasia in the lung airways in a lipopolysaccharide (LPS)-induced lung inflammation murine model while promoting club cell proliferation in a Yap (Yap1)-dependent manner. Additionally, we showed that deleting Mst1/2 is sufficient to suppress p53 deficiency-mediated goblet cell metaplasia. Finally, single-cell RNA-sequencing analysis revealed downregulation of YAP and p53 signaling in goblet cells in human airways. These findings underscore the important role of Hippo and p53 signaling in regulating airway mucous metaplasia.

Hippo cooperates with p53 to regulate lung airway mucous cell metaplasia.

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease with high mortality and limited treatment options. While single-dose bleomycin-induced models are commonly used to investigate the pathogenesis of IPF, they fail to adequately replicate the complex pathological features in human patients, thereby hindering comprehensive investigations. Previous studies utilizing repetitive bleomycin injections have demonstrated a closer resemblance to human IPF pathology; however, the time- and resource-intensive nature of this approach presents significant drawbacks. Here, we propose a novel methodology involving twice-repeated oropharyngeal administration of bleomycin in mice, which closely mirrors the pathological manifestations observed in IPF patients. This model exhibited the honeycomb-like cyst formation, fibroblastic foci, bronchiolization of alveolar epithelium, emergence of metaplastic alveolar KRT5 basal cells, and sustainability of these fibrotic phenotypes, thereby providing a robust model for IPF. Our findings establish a more efficient and translatable preclinical platform for investigating IPF pathogenesis and exploring potential therapeutic strategies.

A novel mouse model of pulmonary fibrosis: twice-repeated oropharyngeal bleomycin administration mimicking human pathology.

 Myogenesis is a strictly regulated process driven by signaling pathways activating muscle-specific gene expression. During myogenesis, muscle stem cells exhibit DNA damage response (DDR) features, which are essential for myoblast differentiation and skeletal muscle regeneration. However, the specific roles of DDR-associated proteins in these processes are not yet fully understood.  Gene knockdown and knockout were used in cell and animal models to study RNF138's function in myoblast differentiation and skeletal muscle regeneration. Multi-omics profiling, including transcriptomics and proteomics, was conducted to identify the key proteins regulated by RNF138 in myogenesis. Protein turnover assays were utilized to investigate RNF138's role in APC protein turnover. Immunofluorescence microscopy was performed to confirm the protein colocalization and subcellular localization.  RNF138 expression increases during myoblast differentiation and in regenerating myofibers following muscle injury. Knockdown of RNF138 in C2C12 myoblasts impairs myogenic differentiation and fusion. Additionally, -deficient mice exhibit delayed muscle regeneration following cardiotoxin-induced injury. Multi-omics profiling, including transcriptomics and proteomics, reveals that Wnt/β-catenin signaling, a key driver of myogenic differentiation, is enhanced by RNF138. Mechanistically, RNF138 stabilizes β-catenin and enhances its nuclear localization by facilitating lysosomal degradation of APC, a component of the β-catenin degradation complex responsible for mediating the export of β-catenin from the nucleus to the cytoplasm for further ubiquitin-proteasome degradation.  We reveal a noncanonical role for RNF138, an E3 ubiquitin ligase, as a positive regulator of myoblast differentiation and skeletal muscle regeneration via the Wnt/β-catenin pathway. This finding highlights the noncanonical function of RNF138 beyond its known roles in DDR and other cellular processes. Therefore, RNF138 provides a potential link between DDR and myoblast differentiation, offering new insights into the molecular regulation of muscle regeneration.

Article	Year
Caveolin-1 deficiency protects pancreatic β cells against palmitate-induced dysfunction and apoptosis. Cellular signalling\| PubMed ID: 29596872	2018
analysis identifies neuropilin-1 as a potential therapeutic target for SARS-Cov-2 infected lung cancer patients. Aging\| PubMed ID: 34168096	2021
Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice. Journal of diabetes research\| PubMed ID: 34917687	2021
The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants. Science translational medicine\| PubMed ID: 35579533	2022
LncRNA HHIP-AS1 suppresses lung squamous cell carcinoma by stabilizing HHIP mRNA. Life sciences\| PubMed ID: 36958438	2023
Review: Non-canonical role of Drosha ribonuclease III. International journal of biological macromolecules\| PubMed ID: 37793530	2023
c-Rel is a Novel Oncogene in Lung Squamous Cell Carcinoma Regulating Cell Proliferation and Migration. Journal of Cancer\| PubMed ID: 38495491	2024
YAP Alleviates Pulmonary Fibrosis Through Promoting Alveolar Regeneration via Modulating the Stemness of Alveolar Type 2 Cells. Stem cells and development\| PubMed ID: 39119800	2024
Hippo cooperates with p53 to regulate lung airway mucous cell metaplasia. Disease models & mechanisms\| PubMed ID: 39428818	2024
A novel mouse model of pulmonary fibrosis: twice-repeated oropharyngeal bleomycin administration mimicking human pathology. Biochemistry and cell biology = Biochimie et biologie cellulaire\| PubMed ID: 39878201	2025
RNF138 regulates skeletal muscle differentiation via the Wnt/β-catenin signaling pathway. Theranostics\| PubMed ID: 40225576	2025

Chuwen Lin

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