Article ID: CJ-25-0055
Background: Because lung fibroblasts play a key role in the pathogenesis of pneumonia, and rho-associated coiled-coil containing protein kinase 1 (ROCK1) is a regulator of lung inflammation, this study studied the action of ROCK1 on lung fibroblast functions under pneumonic conditions.
Methods and Results: WI-38 fibroblasts were stimulated with lipopolysaccharide (LPS) in vitro. A mouse model of pneumonia was produced by LPS induction. IP, Co-IP, and protein stability assays were used to confirm the ubiquitin-specific protease 33 (USP33)/ROCK1 relationship. RIP, Me-RIP, and mRNA stability assays were used to validate the methyltransferase-like 3 (METTL3)/ROCK1 relationship. In LPS-inducible WI-38 cells and serum samples of patients with pneumonia, ROCK1, USP33, and METTL3 levels were increased. ROCK1 deficiency attenuated LPS-evoked apoptosis, inflammation, and oxidative stress in WI-38 fibroblasts and BEAS-2B cells, and also diminished macrophage M1 polarization. Mechanistically, USP33 stabilized ROCK1 protein through deubiquitination, and METTL3 stabilized ROCK1 mRNA in an m6A-IGF2BP1-dependent mode. Depletion of USP33 or METTL3 mitigated LPS-evoked WI-38 cell injuries and macrophage M1 polarization by downregulating ROCK1. Moreover, ROCK1 depletion ameliorated LPS-evoked lung injuries in a pneumonia mouse model.
Conclusions: Our findings suggested that ROCK1 upregulation induced by USP33 and METTL3 affected LPS-evoked dysfunction in WI-38 fibroblasts and lung injuries in pneumonic mice, providing promising therapeutic targets for pneumonia.
Pneumonia is a respiratory infection that affects the lungs, leading to inflammation and fluid accumulation in the alveoli. It is a predominant reason of morbidity and mortality globally, particularly among young children, the elderly, and immunocompromised individuals.1,2 The pathological condition is often caused by various pathogens, including bacteria, viruses, and fungi, with Streptococcus pneumoniae being the most common bacterial cause.3–5 Pneumonia poses significant health risks, leading to severe complications such as respiratory failure, sepsis, and even death if not treated promptly and effectively. Current treatment strategies primarily involve antibiotics for bacterial infections, antiviral medications for viral causes, and supportive care to manage symptoms. However, challenges persist due to increasing antibiotic resistance, limited antiviral options, and the complexity of treating co-infections.6 To develop more effective therapeutic approaches, a deeper understanding of the pathophysiological mechanisms underlying pneumonia is essential.
Lung fibroblasts, a type of mesenchymal cell, play a fundamental role in maintaining the structural integrity of the lung.7 Activated fibroblasts secrete various cytokines and chemokines and interact with immune cells.8,9 Moreover, enhanced oxidative stress and aberrant apoptosis are involved in the pathogenesis of pneumonia, affecting the overall prognosis of pneumonia patients.10 However, despite these significant findings, the precise role of fibroblasts in the pathogenesis of pneumonia remains far from fully understood.
Rho-associated coiled-coil containing protein kinase 1 (ROCK1), a serine/threonine kinase, works as a crucial downstream effector of the small GTPase RhoA and is essential for numerous cellular processes.11,12 ROCK1 has been extensively investigated for its role in effectively regulating immune responses and inflammatory processes by influencing the production of pro-inflammatory cytokines and the activation of immune cells.13 Research into inflammation has unveiled that ROCK1 is implicated in various inflammatory disorders, such as acute central nervous system injury and cardiovascular diseases.14,15 Moreover, in acute lung injury, ROCK1 expression is positively linked to lung inflammation.16 Knockdown of ROCK1 can relieve lipopolysaccharide (LPS)-caused lung inflammation by inactivating the NLRP3 inflammasome.17 Nonetheless, the specific action of ROCK1 on lung fibroblasts under pneumonic conditions has not been extensively reported.
Deubiquitination, a post-translational modification, is mediated by deubiquitinating enzymes and involves the removal of ubiquitin molecules from proteins, thus altering their stability and expression.18 Imbalance of deubiquitination has been implicated in the modulating of the lung inflammatory response.19 m6A methylation, the most prevalent form of RNA modification, controls various aspects of RNA metabolism, including mRNA stability.20 Dysregulation of m6A methylation has been shown to affect the expression and function of genes involved in lung inflammation.21
In the current study, our data revealed that ROCK1 depletion can ameliorate LPS-evoked dysfunction in WI-38 fibroblasts and lung injuries in pneumonic mice. Further, this study explored the molecular determinants driving ROCK1 upregulation from 2 aspects: deubiquitinating enzymes (post-translational modification) and m6A modifiers (post-transcriptional modification). Our findings demonstrated that ubiquitin-specific protease 33 (USP33), a well-reported pro-inflammatory deubiquitinating enzyme,22,23 and methyltransferase-like 3 (METTL3), which is involved in inflammatory injuries,24,25 function as upstream regulators in increasing ROCK1 expression.
The human lung fibroblast WI-38 cell line (#IM-H221, Immocell, Xiamen, China) and human THP-1 cell line (#IM-H260, Immocell) were used and cultured as described in the Supplementary Material. Human bronchial epithelial BEAS-2B cells (#IM-H128) were obtained from Immocell. For LPS stimulation, sterile LPS solution was added to complete culture media at a final concentration of 10 µg/mL as described26 and applied for 12 h at 37℃.
Constructs and Cell TransfectionThe in vitro depletion experiments were done using the following small interfering RNA (siRNA) pools (MedChemExpress, Shanghai, China): ROCK1 Human Pre-designed siRNA Set A (si-ROCK1), USP33 Human Pre-designed siRNA Set A (si-USP33), METTL3 Human Pre-designed siRNA Set A (si-METTL3), IGF2BP1 Human Pre-designed siRNA Set A (si-IGF2BP1), or siRNA control (si-NC).
ImmunoblotsAfter the indicated transfection or/and LPS induction, the protein extracts isolated from cultured WI-38 fibroblasts or lung tissues were resolved by SDS-PAGE for western blot using standard methods.27 Following blocking with 5% non-fat milk, the PVDF membranes were subjected to probing with primary antibodies (Supplementary Material). After horseradish peroxide secondary antibody incubation, a Chemiluminescence Kit (Merck Millipore, Darmstadt, Germany) was used for signal development, and the ChemiDoc XRS System (Bio-Rad, Marnes-la-Coquette, France) was used for densitometry analysis.
Cell Viability, Proliferation and Apoptosis AssaysCell viability was assessed by a CCK-8 assay (MedChemExpress); cell proliferation was detected by an EdU assay (Beyotime, Shanghai, China); and cell apoptosis was examined using flow cytometry. Details are given in the Supplementary Material.
ELISA for Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α LevelsA Human or Mouse IL-6 ELISA Kit (Enzyme-linked Biotechnology, Shanghai, China) was used for determining IL-6 levels, and a Human or Mouse TNF-α ELISA Kit (Beyotime) was used to detect TNF-α levels.
Detection of Malondialdehyde (MDA) and Reactive Cxygen Species (ROS) LevelsThe DCFH-DA ROS Assay Kit and MDA Kit (Beyotime) were used to quantify ROS and MDA levels, respectively.
Multicellular Coculture and M1 Macrophage Subset AnalysisTHP-1 cells were induced to differentiate into macrophages with 10 ng/mL phorbol-12-myristate-13-acetate (PMA, Selleck, Shanghai, China) for 24 h. Multicellular coculture systems were composed of THP-1-differentiated macrophages and WI-38 fibroblasts as described in the Supplementary Material.
Bioinformatics AnalysisAll bioinformatics analyses are shown in the Supplementary Material.
Quantitative Polymerase Chain Reaction (PCR) for ROCK1 mRNA AnalysisAfter RNA extraction with the Total RNA Mini Kit (Bio-Rad), reverse transcription PCR was conducted using 200ng of RNA together with the IScript cDNA Synthesis Kit (Bio-Rad) and quantitative PCR using iQTM SYBR® Green.
Analysis of ROCK1 Protein StabilityTo determine the effect of USP33 on ROCK1 stabilization, a protein stability analysis was performed as described in the Supplementary Material under cycloheximide (CHX, Selleck) treatment.
Immunoprecipitation (IP) and Co-IP ExperimentsA commercial IP Assay Kit (Beyotime) was used to perform these experiments with relevant antibodies. Total extractions of treated WI-38 fibroblasts were prepared and subjected to IP assays as described in the Supplementary Material.
RIP and Methylated RIP (Me-RIP) ExperimentsCellular lysates were prepared, and these experiments were performed as described in the Supplementary Material.
Analysis of ROCK1 mRNA StabilityAfter transfection with si-METTL3, si-IGF2BP1, or si-NC, WI-38 fibroblasts were exposed to 5 µg/mL actinomycin D (Act D, Selleck). After 0, 3, and 6 h, total RNA was extracted and subjected to quantitative PCR for the levels of residual ROCK1.
Animal Groups and TreatmentsAnimal experimental protocols were granted approval by the Animal Care and Use Committee of the First Affiliated Hospital of Xinxiang Medical College. The 20 female BALB/c mice (5–7 weeks old, 16–20 g, Vital River Laboratory, Beijing, China) were divided into 4 groups: sham (n=5), LPS (n=5), LPS+Ad-sh-NC (n=5), and LPS+Ad-sh-ROCK1 (n=5). After 21 days, the mice’s lungs were harvested, weighed, and subjected to further analyses. These details and lung injury assessment criteria are given in the Supplementary Material.
H&E, Masson, and TUNEL Staining, and Immunohistochemical (IHC) AssayAfter being fixed with 4% paraformaldehyde, the lungs were subjected to the experiments described in the Supplementary Material.
Human Serum SamplesHuman serum samples were collected from patients diagnosed with pneumonia (n=34) and healthy volunteers (n=29) at the First Affiliated Hospital of Xinxiang Medical College, following protocols approved by the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical College. Written informed consent was given by all participants. Serum aliquots were transferred to cryovials, immediately frozen at −80℃, and stored until analysis.
Statistical AnalysisANOVA (one- or two-way) with Tukey’s or Sidak’s post-hoc tests were used to compare ≥3 groups, and compared 2 groups using an unpaired Student’s t-test. All data were presented as mean±SD (n ≥3). Statistical significance was set at a P value <0.05.
Because it has been demonstrated that ROCK1 is implicated in pulmonary inflammatory injury,17,28 this study studied the action of ROCK1 under pneumonic conditions by examining the phenotypic alterations of LPS-inducible WI-38 cells following ROCK1 manipulation.
ROCK1 protein levels were remarkably increased in LPS-inducible WI-38 cells (Figure 1A). Thus, this current study sought to silence ROCK1 expression using si-ROCK1 in LPS-inducible WI-38 cells to observe ROCK1’s action. Introduction of si-ROCK1 led to a reduction in ROCK1 protein levels in LPS-inducible WI-38 cells (Figure 1A). Strikingly, ROCK1 depletion rescued LPS-driven viability and proliferation defects in WI-38 fibroblasts (Figure 1B,C). Conversely, deficiency of ROCK1 strongly diminished LPS-triggered apoptosis promotion (Figure 1D). With LPS stimulation, the secretion levels of IL-6 and TNF-α increased (Figure 1E,F) and the expression amounts of MDA and ROS elevated (Figure 1G,H) in WI-38 fibroblasts. However, silencing ROCK1 significantly decreased the increases induced by LPS in WI-38 fibroblasts (Figure 1E–H), indicating that ROCK1 deficiency can mitigate LPS-induced inflammatory injury in WI-38 fibroblasts.
ROCK1 depletion mitigates LPS-evoked apoptosis, inflammation, and oxidative stress in WI-38 fibroblasts and diminishes macrophage M1 polarization. (A–H) Human WI-38 fibroblasts were stimulated with LPS or vehicle, or WI-38 fibroblasts were introduced with si-ROCK1 or si-NC before LPS stimulation. The influence of ROCK1 protein expression (A), cell viability (B), cell proliferation (C), cell apoptosis (D), IL-6 and TNF-α secretion levels (E,F), and MDA and ROS contents (G,H) were evaluated. (I) THP-1-differentiated macrophages were co-cultured with WI-38 fibroblasts treated as indicated in A–H. The ratio of CD86+ macrophages was tested. Scale bars: 50 µm. *P<0.05.
Fibroblasts are associated with macrophage polarization,29,30 and pro-inflammatory M1 macrophages contribute to progression of pneumonia.31 This study thus analyzed the effect of ROCK1 on macrophage M1 polarization. ROCK1 depletion in LPS-inducible WI-38 cells decreased the ratio of CD86+ cells (Figure 1I). Additionally, depletion of ROCK1 by si-ROCK1 significantly enhanced cell growth, hindered cell apoptosis, reduced IL-6 and TNF-α levels, and decreased ROS production in LPS-inducible bronchial epithelial BEAS-2B cells (Supplementary Figure 1A–G). Moreover, incubation of ROCK1-depleted BEAS-2B cells under LPS exposure reduced the ratio of CD86+ cells (Supplementary Figure 1H).
Stabilization of ROCK1 by USP33 Through DeubiquitinationThe ubiquitin-proteasome system, as an inflammatory regulator,32 plays a pivotal role in regulating the degradation of ROCK1.33 To uncover the mechanism driving the ROCK1 increase in LPS-inducible WI-38 cells, this current study identified a related deubiquitinating enzyme. The publicly available Ubibrowser web showed that USP33, a well-reported pro-inflammatory factor,22,23 had the potential to modulate the ROCK1 protein (Figure 2A). USP33 protein levels were elevated in LPS-inducible WI-38 cells, and si-USP33 strongly reduced USP33 protein expression in WI-38 cells treated with LPS (Figure 2B). Although USP33 depletion did not cause reduction of ROCK1 mRNA (Figure 2C), it did, however, decreased ROCK1 protein levels in WI-38 fibroblasts (Figure 2D). Moreover, the ubiquitin-proteasome inhibitor MG132 markedly counteracted USP33 knockdown-imposed ROCK1 protein reduction (Figure 2D). To investigate whether USP33 affected the stability of the ROCK1 protein, this current study blocked protein synthesis with cycloheximide (CHX) in WI-38 cells and confirmed that a deficiency of USP33 weakened the stabilization of ROCK1 protein (Figure 2E). Co-IP assays also validated the interaction between USP33 and ROCK1 in WI-38 cells (Figure 2F). More interestingly, knockdown of USP33 led to an elevation in the level of ubiquitinated ROCK1 protein (Figure 2G). Taken together, these results demonstrated that USP33 enhanced the stability of the ROCK1 protein through deubiquitination.
USP33 stabilizes ROCK1 protein through deubiquitination. (A) The Ubibrowser tool predicted the potential USP33/ROCK1 relationship. (B) WI-38 fibroblasts were stimulated with LPS or vehicle, or WI-38 fibroblasts were introduced with si-USP33 or si-NC before LPS stimulation, followed by the assessment of USP33 protein expression. (C) ROCK1 mRNA in WI-38 cells transfected with si-NC or si-USP33. (D) ROCK1 protein in si-NC- or si-USP33-introduced WI-38 cells with or without MG132 exposure. (E) Quantification of ROCK1 protein in transfected WI-38 cells under cycloheximide (CHX). (F) Immunoprecipitations of cell lysates were performed using the anti-USP33 or anti-ROCK1 antibody. (G) IP experiments with cell lysates using the anti-ROCK1 antibody. *P<0.05.
Effect of USP33 Depletion on LPS-Evoked WI-38 Cell Inflammatory Injuries and Macrophage M1 Polarization
The current study next evaluated whether USP33 affects WI-38 fibroblast functions under LPS via ROCK1. This study used OE-ROCK1 to elevate ROCK1 expression and its efficacy was confirmed (Figure 3A). In LPS-inducible WI-38 cells, USP33 silencing significantly enhanced cell viability (Figure 3B) and proliferation (Figure 3C), but strongly reduced cell apoptosis (Figure 3D). However, these effects of USP33 depletion were partially reversed by increased ROCK1 (Figure 3B–D). Moreover, depletion of USP33 in LPS-inducible WI-38 cells led to downregulation of IL-6 expression, TNF-α secretion, and the levels of MDA and ROS, all of which were strongly abrogated by elevated expression of ROCK1 (Figure 3E–H). When THP-1-differentiated macrophages were cocultured with LPS-inducible WI-38 cells with the indicated transfection, USP33 silencing attenuated macrophage M1 polarization, and this effect was dramatically abolished by the addition of OE-ROCK1 (Figure 3I).
USP33 affects the functions of LPS-inducible WI-38 fibroblasts by ROCK1. (A) Quantification of ROCK1 protein in transfected WI-38 fibroblasts. (B–H) WI-38 fibroblasts were stimulated with LPS or vehicle, or WI-38 fibroblasts were transfected with si-USP33, si-USP33+OE-ROCK1, or si-NC prior to LPS exposure. Effects of cell viability (B), cell proliferation (C), cell apoptosis (D), IL-6 and TNF-α secretion levels (E,F), and MDA and ROS levels (G,H). Scale bar: 50 µm. (I) THP-1-differentiated macrophages were cocultured with WI-38 fibroblasts treated as indicated in B–H, followed by determination of the ratio of CD86+ macrophages. *P<0.05.
Effect of METTL3 on m6A Methylation and Stability of ROCK1 mRNA in the IGF2BP1-Dependent Mode
Dysregulation of METTL3-mediated m6A modification is commonly observed during inflammatory injuries.24,25 Of interest, the ENCORI algorithm predicted the potential relationship between METTL3 and ROCK1 (Figure 4A). Several computational methods (RMbase, RMvar, and SRAMP) predicted the putative m6A methylation modification within ROCK1 mRNA (Figure 4B–D). Therefore, this study explored whether METTL3 could mediate the m6A modification of ROCK1 mRNA in WI-38 fibroblasts. LPS-inducible WI-38 cells exhibited increased levels of METTL3 protein (Figure 4E). With si-METTL3 transfection, the levels of METTL3 protein in LPS-inducible WI-38 cells decreased (Figure 4E). In WI-38 fibroblasts, depletion of METTL3 not only decreased the mRNA expression of ROCK1 (Figure 4F), but also downregulated ROCK1 protein levels (Figure 4G). Furthermore, MeRIP experiments showed that the m6A modification of ROCK1 mRNA existed in WI-38 cells, as demonstrated by massive enrichment of ROCK1 mRNA in m6A-related precipitates, and METTL3 depletion decreased m6A-modified ROCK1 mRNA levels (Figure 4H). RIP assays verified the interaction between METTL3 and ROCK1 in WI-38 cells (Figure 4I). Act D treatment revealed that METTL3 silencing significantly weakened the stability of ROCK1 mRNA (Figure 4J).
METTL3 enhances the m6A methylation and stability of ROCK1 mRNA. (A) ENCORI algorithm predicted the potential METTL3/ROCK1 relationship. (B–D) RMbase (B), RMvar (C), and SRAMP (D) predicted the putative m6A methylation within ROCK1 mRNA. (E) METTL3 protein in treated WI-38 cells. (F,G) Levels of ROCK1 in transfected WI-38 cells. (H) MeRIP experiments with cell lysates using the anti-m6A antibody and quantitative PCR of ROCK1 mRNA enrichment levels in precipitates. (I) RIP experiments with cell lysates using the anti-METTL3 or anti-IgG antibody and quantification of ROCK1 mRNA enrichment levels in precipitates. (J) WI-38 cells transfected with si-METTL3 or si-NC were subjected to actinomycin D (Act D) treatment and checked for residual ROCK1 mRNA levels. *P<0.05.
The effect of METTL3-driven m6A modification is mediated by m6A readers, which influence the stability of mRNA.34 The ENCORI algorithm revealed a potential relationship between ROCK1 and IGF2BP1 (Figure 5A), a crucial m6A reader associated with the inflammatory response.35,36 Deficiency of IGF2BP1 by si-IGF2BP1 transfection (Figure 5B) resulted in decreased levels of ROCK1 in WI-38 cells (Figure 5C,D). RIP assays demonstrated the ROCK1/IGF2BP1 relationship, and METTL3 depletion diminished their relationship (Figure 5E). Additionally, IGF2BP1 deficiency significantly weakened ROCK1 mRNA stabilization in WI-38 cells under Act D treatment (Figure 5F). Collectively, METTL3 stabilized ROCK1 mRNA through an m6A-IGF2BP1-dependent mechanism.
IGF2BP1 enhances the stability of ROCK1 mRNA. (A) ENCORI algorithm predicted the potential relationship between IGF2BP1 and ROCK1. (B–D) IGF2BP1 protein expression (B), ROCK1 mRNA levels (C), and ROCK1 protein expression (D) in WI-38 cells transfected with si-IGF2BP1 or si-NC. (E) RIP experiments with cell lysates using the anti-IGF2BP1 or anti-IgG antibody. (F) Transfected WI-38 cells were exposed to Act D, followed by detection of residual ROCK1 mRNA levels. *P<0.05.
Effect of METTL3 on LPS-Triggered WI-38 Cell Inflammatory Injuries and Macrophage M1 Polarization via ROCK1
Because METTL3 can enhance inflammatory injuries in LPS-stimulated WI-38 cells,37 this current study also examined the contribution of ROCK1 in the effect of METTL3. Strikingly, METTL3 knockdown in LPS-inducible WI-38 cells promoted cell viability and proliferation, but impeded apoptosis, all of which were reversed by increased ROCK1 levels (Figure 6A–C). Moreover, METTL3 depletion decreased the levels of IL-6, TNF-α, MDA, and ROS in LPS-inducible WI-38 cells; however, these alterations were significantly abrogated by ROCK1 increase (Figure 6D–G). Further, coculture of THP-1-differentiated macrophages with LPS-inducible WI-38 cells with METTL3 silencing reduced the ratio of CD86+ macrophages, and the change was remarkably counteracted by increased ROCK1 levels (Figure 6H).
METTL3 affects LPS-triggered WI-38 cell inflammatory injuries and macrophage M1 polarization via ROCK1. (A–G) WI-38 fibroblasts were transfected with or without si-METTL3, si-METTL3+OE-ROCK1, or si-NC before LPS induction. The influences of cell viability (A), cell proliferation (B), cell apoptosis (C), IL-6 and TNF-α secretion levels (D,E), and MDA and ROS levels (F,G) were detected. Scale bar: 50 µm. (H) THP-1-differentiated macrophages were cocultured with WI-38 fibroblasts treated as indicated in A–G, followed by determination of the ratio of CD86+ macrophages. *P<0.05.
Effect of Deficiency of ROCK1 on LPS-Evoked Lung Injuries in a Mouse Model of Pneumonia
To further validate the function of ROCK1 in vivo, the current study subsequently developed a mouse model of pneumonia accompanied by administration of Ad-sh-ROCK1. The lung tissues of sham mice maintained their normal structure with distinct alveolar spaces, whereas the lungs of LPS-induced pneumonia mice displayed thickened alveolar septa, epithelial cell shedding, and enhanced inflammatory cell infiltration (Figure 7A). Masson staining also showed that the lungs of LPS-induced pneumonia mice exhibited increased collagen fiber formation (Figure 7A). Moreover, the pneumonic mice had higher lung injury scores and elevated lung W/D ratios compared with the sham mice (Figure 7B,C). TUNEL staining confirmed that the pulmonary cells of pneumonic mice exhibited enhanced apoptotic ratios (Figure 7D). Intriguingly, injection of Ad-sh-ROCK1 significantly alleviated these pathological changes, collagen formation, lung injury, and cell apoptosis (Figure 7A–D). IHC and immunoblot assays of the mice’s lungs revealed increased levels of ROCK1 in the LPS-induced pneumonic mice, in which injection of Ad-sh-ROCK1 strongly decreased ROCK1 expression (Figure 7E,F). In addition, IL-6 and TNF-α levels were elevated in the lungs of the pneumonic mice and these were markedly decreased following Ad-sh-ROCK1 injection (Figure 7G,H). Thus, ROCK1 depletion ameliorated LPS-evoked lung injuries in the pneumonic mice.
Depletion of ROCK1 ameliorates LPS-evoked lung injuries in pneumonic mice. (A) H&E staining and Masson staining of lung sections. (B,C) Lung injury score and lung wet weight (W)/dry weight (D) ratio of mouse lungs. (D) TUNEL staining of lung tissues. (E) IHC assay of ROCK1 in mouse lungs. (F) Immunoblots of ROCK1 protein in mouse lungs. (G,H) Levels of IL-6 and TNF-α in the suspension of murine lungs. Scale bars: 50 µm. *P<0.05.
Silencing of ROCK1 can exert anti-inflammatory effects in in microglia in LPS-triggered neuroinflammation.14 ROCK1 induces a long-lived inflammatory response in inflammatory bowel disease,38 and dysregulation of ROCK1 is related to high glucose-evoked inflammation in human retinal pigment epithelial cells.39 Moreover, there are reports highlighting the promoting effect of ROCK1 in lung inflammation induced by various stimuli.16,17 Our current study revealed that ROCK1 depletion attenuates LPS-evoked apoptosis, pro-inflammatory cytokine generation, and oxidative stress in WI-38 fibroblasts and BEAS-2B cells in vitro. Furthermore, in a mouse model of pneumonia, ROCK1 deficiency ameliorated LPS-triggered lung injuries. Pro-inflammatory M1 macrophages play a pivotal role in both the inflammatory response and the progression of pneumonia.31 Modulation of fibroblasts in macrophage polarization has been shown,29 and interestingly, our study demonstrated that ROCK1 depletion in LPS-inducible WI-38 fibroblasts and BEAS-2B cells can suppress macrophage M1 polarization. Given its critical role in modulating inflammation and lung injuries, ROCK1 represents a promising therapeutic target for pneumonia.
Recent research has highlighted the involvement of USP33, a key deubiquitinating enzyme, in modulating diverse inflammatory conditions, including acute pancreatitis.22 Moreover, USP33 has been reported to be closely related to lung inflammatory disorders.40,41 A recent study highlighted that depletion of USP33 had antiviral and anti-inflammatory effects in SARS-CoV-2-infected lung tissues.23 Our data have established that USP33 stabilized ROCK1 protein through deubiquitination in WI-38 fibroblasts. More intriguingly, our study showed that USP33 depletion mitigated LPS-evoked WI-38 cell inflammatory injuries and macrophage M1 polarization by downregulating ROCK1. These findings uncover a new mechanism by which USP33 affects the functions of LPS-inducible WI-38 fibroblasts through ROCK1 modulation. However, the specific ubiquitination sites on the ROCK1 protein and the functional domains of USP33 that regulate this process are yet to be elucidated. Further research is needed to fully determine the mechanisms underlying the interaction between USP33 and ROCK1.
METTL3, an RNA methyltransferase responsible for m6A methylation, has been increasingly recognized for its role in modulating gene expression and cellular processes in various inflammatory diseases, including renal inflammation, intestinal inflammation, and neuroinflammation.24,42,43 METTL3 has emerged as a molecule of great interest in lung inflammation. For example, its expression and m6A-modified mRNA levels are upregulated in the lung tissues of acute lung injury mice, and METTL3 silencing reduces lung inflammation.44 METTL3 has also been found to enhance cell apoptosis and inflammation in LPS-inducible WI-38 fibroblasts.37 Our current investigation discovered that METTL3 enhanced the m6A methylation and stability of ROCK1 mRNA in an IGF2BP1-dependent way. Our rescue experiments demonstrated that METTL3 affected LPS-triggered inflammatory injuries in WI-38 fibroblasts and macrophage M1 polarization via ROCK1. These findings highlight a novel mechanism by which the METTL3/IGF2BP1 axis regulates lung inflammation through ROCK1. However, the specific m6A modification sites on ROCK1 mRNA mediated by the METTL3/IGF2BP1 cascade remain to be elucidated and will be explored in future work.
Interestingly, the clinical data from patients with pneumonia showed high mRNA levels of ROCK1, USP33, and METTL3 in their serum samples (n=34) compared with healthy controls (n=29) (Supplementary Figure 2A–C). Future studies will expand expression analyses in serum samples to include a larger cohort of patients with pneumonia to elucidate whether increased levels of ROCK1, USP33, and METTL3 are associated with disease severity. Additionally, systemic delivery of adenoviral vectors often results in predominant liver transduction. Thus, lack of tail vein injection of adenovirus expressing sh-ROCK1 into the pneumonic mice is the biggest limitation of our current study. This study focused on the pulmonary effects of sh-ROCK1 adenovirus in the context of LPS-induced pneumonia, but acknowledge the predominant hepatic tropism of intravenously administered adenovirus. By packaging the sh-ROCK1 adenovirus with a CMV promoter, requiring higher viral titers, injecting virus at optimized doses, this study demonstrated that tail vein injection of sh-ROCK1 adenovirus significantly reduced ROCK1 expression in the lung tissues of the pneumonic mice, confirming that the virus can also disseminate to other organs, including the lungs, albeit at lower efficiencies. Direct lung-targeted delivery methods, such as intratracheal instillation, might have provided more localized knockdown but were not utilized in this particular study design, but our teams expect to perform it in future work.
In summary, our current study demonstrated that ROCK1 depletion ameliorated LPS-evoked dysfunction in WI-38 fibroblasts, BEAS-2B cells and lung injuries in pneumonic mice. USP33 and METTL3 are 2 upstream regulators driving ROCK1 upregulation under pneumonic conditions. Our study results suggest that targeting ROCK1 or its regulatory cascade may be useful for the treatment and prevention of pneumonia.
(1) USP33 stabilizes ROCK1 protein.
(2) METTL3 stabilizes ROCK1 mRNA in an m6A-IGF2BP1-dependent way.
(3) ROCK1 affects lung injuries in pneumonia mice.
None.
This study was supported by 2024 Henan Province Medical Science and technology research plan joint construction project [Award Number: LHGI20240469].
The authors declare that they have no conflicts of interest.
Y.L. and Y.Y. designed and performed the research; S.Z., J.Z. and F.H. analyzed the data; J.F. and C.Z. wrote the manuscript. All authors read and approved the final manuscript.
Not applicable.
Animal studies were performed in compliance with the ARRIVE guidelines and the Basel Declaration. All animals received humane care according to the National Institutes of Health (USA) guidelines.
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https://doi.org/10.1253/circj.CJ-25-0055
