Rho-associated Coiled-coil Containing Protein Kinase Research Articles

Galangin Regulates Astrocyte Phenotypes to Ameliorate Cerebral Ischemia-reperfusion Injury by Inhibiting the RhoA/ROCK/LIMK Pathway.

This study aimed to explore whether Galangin (Gal) could improve cerebral Ischemia- reperfusion (I/R) injury by regulating astrocytes, and clarify its potential molecular mechanism. An I/R injury model of rats was established using the Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) method, followed by the administration of Gal (25, 50, 100 mg/kg) via gavage for 14 consecutive days. Besides, astrocytes were isolated from the rats to construct an Oxygen-Glucose Deprivation/Re-oxygenation (OGD/R) cell model, with treatments of Gal or the Ras homolog gene family member A (RhoA)/Rho-associated Coiled-coil containing protein Kinase (ROCK) inhibitor Y-27632. Subsequently, the severity of nerve injury was assessed using the modified Neurological Severity Score (mNSS) test; behavioral disorders in I/R rats were observed through the open field and ladder-climbing tests. Pathological damages and neuron survival in the peri-infarct zone were examined by hematoxylin and eosin staining and NeuN staining, respectively. Additionally, immunofluorescence staining was employed to determine astrocyte polarization and TUNEL staining was carried out to measure the level of cell apoptosis; also, western blot was performed to detect the expression of proteins related to the RhoA/ROCK/LIM domain Kinase (LIMK) pathway. Gal significantly ameliorated the neurological and motor dysfunctions caused by I/R in rats, reduced pathological damage in the peri-infarct zone, and promoted neuronal survival. Additionally, Gal increased the number of A2 astrocytes, while it decreased the number of A1 astrocytes. In vitro experiments revealed that the effect of Gal was consistent with that of Y-27632. Additionally, Gal significantly enhanced the survival of OGD/R cells, increased the number of A2 astrocytes, and inhibited the expression of proteins associated with the RhoA/ROCK pathway. Gal could reduce the level of apoptosis, promote the polarization of A2 astrocytes, and improve cerebral I/R injury, and its mechanism may be related to the inhibition of the RhoA/ROCK pathway.

H2S protects rat cerebral ischemia-reperfusion injury by inhibiting expression and activation of hippocampal ROCK2 at the Thr436 and Ser575 sites

BackgroundH2S is an endogenous gas signal molecule, which protects cerebral ischemia/reperfusion (I/R) injury by phosphorylating rho-associated coiled coil-containing protein kinase 2 (ROCK2) at Tyr722, and inhibiting ROCK2 protein expression and activities. We previously reported that H2S protected rat neurons from hypoxia/reoxygenation injury in vitro through inhibiting phosphorylation of ROCK2 at Thr436 and Ser575, but it is unclear whether these two sites are involved in protection of H2S against cerebral I/R injury. MethodRats transfected with wild-type and mutant eukaryotic plasmids of ROCK2 in hippocampus were used to establish I/R model by ligating bilateral common carotid artery. Rat behavioral deficit was detected by water maze assay, and ROCK2, lactate dehydrogenase (LDH), nerve-specific enolase (NSE) and reactive oxygen species (ROS) were determined by ELISA. ROCK2 expressions was examined by western-blot assay, and bcl-2 and Bax mRNAs were examined by RT-qPCR. ResultsNaHS (4.8mg/kg) significantly inhibited the I/R-increased serum LDH, NSE and ROS in the ROCK2wild-pEGFP-N1-transfected rats, but had no obvious effect in the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected rats; inhibitions of NaHS on the I/R-increased escape latency and the I/R-decreased percentage of target quadrant distance to total distance were markedly attenuated or abolished in the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected rats compared with those in the ROCK2wild-pEGFP-N1-transfected rats; NaHS obviously inhibited the I/R-increased hippocampal ROCK2 and GFP-ROCK2 proteins, Bax mRNA, and ROCK2 activity, as well as the I/R-decreased hippocampal bcl-2 mRNA in the hippocampus of the ROCK2wild-pEGFP-N1-transfected rats, but had no significant effect in the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected rats. ConclusionH2S protects cerebral I/R injury in rats by inhibiting expression and activation of hippocampal ROCK2 via the Thr436 and Ser575 sites.

Rho-associated, coiled-coil-containing protein kinase 2 regulates expression of mineralocorticoid receptor to mediate sodium reabsorption in mice

The mineralocorticoid receptor (MR) is a member of the nuclear receptor family that was initially identified to regulate blood pressure through its ability to modulate kidney sodium handling in response to aldosterone. MR can be regulated by signals other than aldosterone; however, the detailed mechanisms remain to be elucidated. We found that MR is controlled in a Rho-associated coiled-coil-containing protein kinase 2 (ROCK2)-dependent manner. Mice with a specific deletion of ROCK2 in the kidney tubules showed decreased MR expression levels and increased urinary excretion of sodium. Mechanistically, signal transducer and activator of transcription 3 (STAT3) is a key molecule that mediates MR expression in these mice. This study highlights an important role of tubular ROCK2 in electrolyte homeostasis.

Hydroxysafflor yellow A, a natural food pigment, ameliorates atherosclerosis in ApoE−/− mice by inhibiting the SphK1/S1P/S1PR3 pathway

AbstractAtherosclerosis (AS) is the pathologic basis of many cardiovascular diseases (CVDs). Hydroxysafflor yellow A (HSYA) is a valuable natural food pigment that has been reported to have significant health‐promoting abilities. However, the anti‐AS efficacy and mechanisms of HSYA have not yet been characterized. Here, we found that treatment of apolipoprotein A (ApoE) knockout (ApoE−/−) mice with HSYA markedly ameliorated atherosclerosis evidenced by decreased levels of lipids, sphingosine‐1‐phosphate (S1P), inflammatory factors, oxidative stress, vascular endothelial permeability, and endothelial damage. Moreover, mechanistic studies revealed that HSYA treatment downregulated the expression of aortic sphingosine kinase 1 (SphK1), sphingosine‐1‐phosphate receptor 3 (S1PR3), Ras homolog family member A (RhoA), Rho‐associated coiled‐coil containing protein kinase (ROCK), and filamentous actin (F‐actin). The results of administration with HSYA reversed the effects of SphK1 agonist and S1PR3 agonist on oxidized low‐density lipoprotein (ox‐LDL)‐induced vascular endothelial cell migration ability and F‐actin expression, and decreased RhoA/ROCK protein expression further confirmed the conclusion that HSYA reduced vascular endothelial permeability by modulating the SphK1/S1P/S1PR3/RhoA/ROCK signaling pathway, thereby exerting anti‐atherosclerotic effects. Overall, this study indicated that HSYA might be a therapeutic candidate for the treatment of AS.

GLYAT suppresses liver cancer and clear cell renal cell carcinoma progression by downregulating ROCK1 expression.

The liver and kidney are important metabolic organs in the body and common sites of tumor occurrence. Glycine-N-acyltransferase (GLYAT) is primarily expressed in the liver and kidney and downregulated in several tumors. But its specific functions and molecular mechanisms in liver cancer and clear cell renal cell carcinoma (ccRCC) have not yet been fully elucidated. The aim of this study was to explore the role and clinical significance of GLYAT in liver cancer and ccRCC. This study used proteomics technology to identify differentially expressed proteins in liver cancer. Western blot and immunohistochemistry (IHC) were used to analyze the protein expression pattern of GLYAT. assays were performed in liver cancer and ccRCC cells. Xenograft models in nude mice were used to confirm the roles of GLYAT in liver cancer. Moreover, the downstream regulatory proteins of GLYAT were identified by proteomics. GLYAT was lowly expressed in liver cancer and ccRCC. Immunofluorescence staining indicated that GLYAT was mainly expressed in the cytoplasm, particularly the mitochondria. Kaplan-Meier curves showed that the low protein expression of GLYAT was correlated with a poor prognosis in liver cancer and ccRCC patients. Moreover, GLYAT expression was associated with several clinical parameters in liver cancer. Cell experiments showed that the overexpression of GLYAT inhibited cell proliferation and migration abilities; however, interfering GLYAT protein expression rescued these abilities in GLYAT overexpression (GLYAT-OE) cells. In vivo assays confirmed the tumor-suppressor function of GLYAT in liver cancer. Moreover, our research showed that GLYAT downregulated Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). Our study showed that GLYAT is lowly expressed in liver cancer and ccRCC, emphasizing its prognostic significance. It also showed that GLYAT inhibits the progression of liver cancer and ccRCC by downregulating ROCK1.

Nuclear translocation of nucleotide enzyme Phosphoglucomutase 2 governs DNA damage response and anti-tumor immunity

Targeting nucleotide enzymes emerges as a promising avenue for impeding tumor proliferation and fortifying anti-tumor immunogenicity. The non-canonical role of nucleotide enzymes remains poorly understood. In this study, we have identified that Phosphoglucomutase 2 (PGM2) rapidly accumulates at the DNA damage site to govern the DNA damage response mediated by the phosphorylation at Serine 165 and by forming a complex with Rho-associated coiled-coil-containing protein kinase 2 (ROCK2). Silencing PGM2 in Glioblastoma Multiforme (GBM) cells heightens DNA damage in vitro and enhances the sensitivity of temozolomide (TMZ) treatment by activating anti-tumor immunity in vivo. Furthermore, we demonstrate that pharmacological inhibition of ROCK2 synergistically complements TMZ treatment and pembrolizumab (PD-L1) checkpoint immunotherapy, augmenting anti-tumor immunity. This study reveals the non-canonical role of the nucleotide enzyme PGM2 in the regulation of DNA damage response and anti-tumor immunity, with implications for the development of therapeutic approaches in cancer treatment.

RhoA/ROCK-TAZ Axis regulates bone formation within calvarial trans-sutural distraction osteogenesis

BackgroundCraniofacial skeletal deformities can be addressed by applying tensile force to sutures to prompt sutural bone formation. The intricate process of mechanical modulation in craniofacial sutures involves complex biomechanical signal transduction. The small GTPase Ras homolog gene family member A (RhoA) functions as a key mechanotransduction protein, orchestrating the dynamic assembly of the cytoskeleton by activating the Rho-associated coiled-coil containing protein kinase (ROCK). Transcriptional coactivator with PDZ-binding motif (TAZ) serves as a crucial mediator in the regulation of genes and the orchestration of biological functions within the mechanotransduction signaling pathway. However, the role of RhoA/ROCK-TAZ in trans-sutural distraction osteogenesis has not been reported. MethodsWe utilized pre-osteoblast-specific RhoA deletion mice to establish an in vivo calvarial trans-sutural distraction model and an in vitro mechanical stretch model for pre-osteoblasts isolated from neonatal mice. Micro-CT and histological staining were utilized to detect the formation of new bone in the sagittal suture of the skull as well as the activation of RhoA, Osterix and TAZ. The activation of ROCK-limk-cofilin and the nuclear translocation of TAZ in pre-osteoblasts under mechanical tension were detected through Western blot, qRT-PCR, and immunofluorescence. ResultsThe osteogenic differentiation of pre-osteoblasts was facilitated by mechanical tension through the activation of RhoA and Rho-associated kinase (ROCK), while ablation of RhoA impaired osteogenesis by inhibiting pre-osteoblast differentiation after suture expansion. Furthermore, inhibiting RhoA expression could block tensile-stimulated nuclear translocation of TAZ by preventing F-actin assembly through ROCK-LIM-domain kinase (LIMK)-cofilin pathway. In addition, the TAZ agonist TM-25659 could attenuate impaired osteogenesis caused by ablation of RhoA in pre-osteoblasts by increasing TAZ nuclear accumulation. ConclusionsThis study demonstrates that mechanical stretching promotes the osteogenic differentiation of pre-osteoblasts in trans-sutural distraction osteogenesis, and this process is mediated by the RhoA/ROCK-TAZ signaling axis. Overall, our results may provide an insight for potential treatment strategies for craniosynostosis patients through trans-sutural distraction osteogenesis.

ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway

BackgroundDysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity. MethodsThe biological function of ROCK1 was analyzed in vitro by establishing a silenced cell model. Coimmunoprecipitation confirmed the direct binding between ROCK1 and c-MYC, and a luciferase reporter assay revealed the binding of c-MYC to the promoter of the PFKFB3 gene. These results were verified in animal experiments. ResultsROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with a poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased gemcitabine (GEM) sensitivity in vivo and in vitro. ConclusionsROCK1 promotes glycolytic activity in PC cells and promotes PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth in vivo and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC.

Protective effects of Y-27632 against cisplatin-induced ototoxicity: A zebrafish model Y-27632 and cisplatin-induced ototoxicity

Cisplatin is an effective chemotherapy agent against various solid malignancies; however, it is associated with irreversible bilateral sensorineural hearing loss, emphasizing the need for drug development to prevent this complication, with the current options being very limited. Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine protein kinase involved in various cellular processes, including apoptosis regulation. In this study, we used a transgenic zebrafish model (Brn3C: EGFP) in which hair cells within neuromasts are observed in green under fluorescent microscopy without the need for staining. Zebrafish larvae were exposed to cisplatin alone or in combination with various concentrations of Y-27632, a potent ROCK inhibitor. Hair cell counts, apoptosis assessments using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, FM1-43FX labeling assay and behavioral analyses (startle response and rheotaxis) were performed to evaluate the protective effects of Y-27632 against cisplatin-induced ototoxicity. Cisplatin treatment reduced the number of hair cells in neuromasts, induced apoptosis, and impaired zebrafish larval behaviors. Y-27632 demonstrated a dose-dependent protective effect against cisplatin-induced hair cell loss and apoptosis. These findings suggest that Y-27632, as a ROCK inhibitor, mitigates cisplatin-induced hair cell loss and associated ototoxicity in zebrafish.

The Specific ROCK2 Inhibitor KD025 Alleviates Glycolysis through Modulating STAT3-, CSTA- and S1PR3-Linked Signaling in Human Trabecular Meshwork Cells.

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated. Gene ontology analysis revealed that DEGs were more frequently related to the plasma membrane, extracellular components and integral cellular components among cellular components, and related to signaling receptor binding and activity and protein heterodimerization activity among molecular functions. Ingenuity Pathway Analysis (IPA) revealed that the detected DEGs were associated with basic cellular biological and physiological properties, including cellular movement, development, growth, proliferation, signaling and interaction, all of which are associated with cellular metabolism. Furthermore, the upstream regulator analysis and causal network analysis estimated IL-6, STAT3, CSTA and S1PR3 as possible regulators. Current findings herein indicate that ROCK2 mediates the IL-6/STAT3-, CSTA- and S1PR3-linked signaling related to basic biological activities such as glycolysis in HTM cells.

Low-level PM2.5 induces the occurrence of early pulmonary injury by regulating circ_0092363

The significant correlation between particulate matter with aerodynamic diameters of ≤ 2.5 µm (PM2.5) and the high morbidity and mortality of respiratory diseases has become the consensus of the research. Epidemiological studies have clearly pointed out that there is no safe concentration of PM2.5, and mechanism studies have also shown that exposure to PM2.5 will first cause pulmonary inflammation. Therefore, the purpose of this study is to explore the mechanism of early lung injury induced by low-level PM2.5 from the perspective of epigenetics. Based on the previous results of population samples, combined with an in vitro/vivo exposure model of PM2.5, it was found that low-level PM2.5 promoted the transport of circ_0092363 from intracellular to extracellular spaces.The decreased expression of intracellular circ_0092363 resulted in reduced absorption of miR-31-5p, leading to inhibition of Rho associated coiled-coil containing protein kinase 1 (ROCK1) and the subsequent abnormal expression of tight junction proteins such as Zonula occludens protein 1 (ZO-1) and Claudin-1, ultimately inducing the occurrence of early pulmonary injury. Furthermore, this study innovatively introduced organoid technology and conducted a preliminary exploration for a study of the relationship among environmental exposure genomics, epigenetics and disease genomics in organoids. The role of circ_0092363 in early pulmonary injury induced by low-level PM2.5 was elucidated, and its value as a potential diagnostic biomarker was confirmed.

Deletion of podocyte Rho-associated, coiled-coil-containing protein kinase 2 protects mice from focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) shares podocyte damage as an essential pathological finding. Several mechanisms underlying podocyte injury have been proposed, but many important questions remain. Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) is a serine/threonine kinase responsible for a wide array of cellular functions. We found that ROCK2 is activated in podocytes of adriamycin (ADR)-induced FSGS mice and cultured podocytes stimulated with ADR. Conditional knockout mice in which the ROCK2 gene was selectively disrupted in podocytes (PR2KO) were resistant to albuminuria, glomerular sclerosis, and podocyte damage induced by ADR injection. In addition, pharmacological intervention for ROCK2 significantly ameliorated podocyte loss and kidney sclerosis in a murine model of FSGS by abrogating profibrotic factors. RNA sequencing of podocytes treated with a ROCK2 inhibitor proved that ROCK2 is a cyclic nucleotide signaling pathway regulator. Our study highlights the potential utility of ROCK2 inhibition as a therapeutic option for FSGS.

Abstract 720: Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer

Abstract Zelasudil (RXC007) is an orally available, highly selective small molecule inhibitor of Rho-associated coiled-coil containing protein kinase 2 (ROCK2), which has demonstrated pre-clinical anti-fibrotic efficacy and is currently being tested in a Phase 2a clinical trial for the treatment of idiopathic pulmonary fibrosis (IPF) (NCT05570058). Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and therapeutically challenging disease with a dense and highly fibrotic tumor microenvironment which may limit efficacy of conventional therapies. Here, we investigate the benefit of combining an anti-fibrotic, RXC007, with either standard of care chemotherapy or immunotherapy in mouse models of PDAC.In a fibrotic patient-derived metastatic PDAC orthotopic model, RXC007 (10mg/kg, 50mg/kg and 100mg/kg BID 5d on/2d off PO) in combination with SoC chemotherapy Gemcitabine (100mg/kg IP QW)/Abraxane (30 mg/kg IP QW) improved survival in a dose-dependent manner (all P<0.0001) compared to Gemcitabine/Abraxane alone.In the immunocompetent orthotopic LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of metastatic PDAC, survival was significantly increased (P<0.0001) upon treatment with a combination of RXC007 (50 mg/kg BID 5d on/2d off PO) and anti-PD1 (200 ug BIW) compared to anti-PD1 alone. ROCK2 inhibition elicited positive immunomodulatory effects in KPC pancreatic tumors, with increase of CD8+ (P=0.0031) and CD4+ (P=0.008) T cell infiltrate into the tumor cortex and reduction in immunosuppressive FOXP3+ regulatory T cells at the tumor border. Furthermore, RXC007 monotherapy exhibited anti-fibrotic effects, via a decrease in collagen content and organization, effects associated with CAF reprogramming and reduced intra-tumoral aSMA+ PDPN+ myofibroblast-like CAFs (myCAFs; P=0.0406).This data highlights the potential for treating highly fibrotic tumours with RXC007 in combination with SoC chemotherapy or immunotherapy in order to improve patient outcomes in this aggressive and treatment-refractory cancer. Citation Format: Marina Pajic, Benjamin McLean, Diego Chacon Fajardo, Sean Porazinski, Dannielle H. Upton, Diana Schuhmacher, Aji Istadi, Thomas Cox, Paul Timpson, Daniel J. Wilcock, Katie J. Anderson, Helen Mason, Nicolas E. Guisot, Clifford D. Jones, Caroline Phillips, Richard Armer. Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 720.

Phillygenin prevents osteoclast differentiation and bone loss by targeting RhoA.

Forsythia suspensa tea is a popular traditional Chinese medicine decoction for its healthy and therapeutic benefits. However, its effects in bone metabolism were not clear. In recent study, we uncovered anti-osteoclastogenesis property of Phillygenin (Phi), a compound abundant in Forsythia suspensa leaves, and aimed to investigate the effect and mechanism of Phi on bone metabolism in vivo and in vitro. Lipopolysaccharides-induced murine calvaria osteolysis and ovariectomy-induced bone loss animal models were used to identify the bone-protective effect of Phi in vivo and micro-CT, pQCT, and TRAP staining were applied. We used CCK8, TUNEL, BrdU, and TRAP staining to evaluate the efficacy of Phi on the proliferation and formation of OCs in primary mBMMs. RNA sequence, activity-based protein profiling, molecular docking, G-LISA, and WB were used to inspect the target and underlying mechanism of Phi's actions in mBMMs. We found Phi significantly inhibited bone resorption in vivo and inhibited mBMMs osteoclastogenesis in vitro. Ras homolog gene family member A (RhoA) was identified as the direct target of Phi. It counteracted the effects of RhoA activator and acted as a RhoA inhibitor. By targeting RhoA, Phi modulated Rho-associated coiled-coil containing protein kinase 1 (ROCK1) activity and regulated its downstream NF-κB/NFATc1/c-fos pathway. Furthermore, Phi depressed the disassembling of F-actin ring through cofilin and myosin1a. Our findings provided Phi as a potential option for treating bone loss diseases by targeting RhoA and highlighted the importance of F. suspensa as a preventive approach in bone disorders.

Activation of RhoA/ROCK2 signaling by hypoxia-inducible factor 1α in promoting tumor growth and metastasis in human colon cancer.

Recent evidence strongly suggests the profound role of the tumor microenvironment in cancer development and progression. A hypoxic microenvironment is widely acknowledged to be a typical feature of solid tumors, and altered hypoxia-inducible factor 1α (HIF-1α) expression has been associated with the formation and the progression of many solid tumors; however, the underlying mechanism of this relationship remains obscure. Clinical colorectal cancer tissue samples were collected to detect the differential expression of HIF-1α, Ras homolog family member A (RhoA), and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2). With hypoxic stress, small interfering RNA (siRNA) targeting HIF-1α, lentivirus transfection of RhoA was used to study the mechanism of HIF-1α and RhoA/ROCK2 signaling pathways in the growth and metastasis of colon cancer. According to Cell Counting Kit 8, wound-healing, and Transwell assays, HIF-1α expression activated the RhoA/ROCK2 pathway within colon cancer cell lines, accelerating their growth and expansion. In cells transfected with LV-RhoA, inactivating the RhoA/ROCK2 pathway with the specific inhibitor Y-27632 decreased tumor growth and metastasis under hypoxic conditions, while activating the RhoA/ROCK2 pathway restored these biological properties. The Western blot results showed that the expression levels of pMYPT1, cyclin D1, and MMP2 in the siRNA + LV-RhoA group were also significantly increased compared with those in the siRNA group. For the first time, this study demonstrated that HIF-1α can promote the growth and metastasis of colon cancer via directly affecting RhoA/ROCK2 signaling and thus represents a novel therapeutic target for colon cancer.

Circ_0114428 knockdown inhibits ROCK2 expression to assuage lipopolysaccharide-induced human pulmonary alveolar epithelial cell injury through miR-574-5p

BackgroundSepsis-induced acute lung injury (ALI) accounts for about 40% of ALI, accompanied by alveolar epithelial injury. The study aimed to reveal the role of circular RNA_0114428 (circ_0114428) in sepsis-induced ALI.MethodsHuman pulmonary alveolar epithelial cells (HPAEpiCs) were treated with lipopolysaccharide (LPS) to mimic a sepsis-induced ALI cell model. RNA expression of circ_0114428, miR-574-5p and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) was detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine (EdU) and flow cytometry analysis, respectively. The levels of pro-inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was analyzed by lipid peroxidation Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity detection assays. The interplay among circ_0114428, miR-574-5p and ROCK2 was identified by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assays.ResultsCirc_0114428 and ROCK2 expression were significantly increased, but miR-574-5p was decreased in blood samples from sepsis patients and LPS-stimulated HPAEpiCs. LPS treatment led to decreased cell viability and proliferation and increased cell apoptosis, inflammation and oxidative stress; however, these effects were relieved after circ_0114428 knockdown. Besides, circ_0114428 acted as a miR-574-5p sponge and regulated LPS-treated HPAEpiC disorders through miR-574-5p. Meanwhile, ROCK2 was identified as a miR-574-5p target, and its silencing protected against LPS-induced cell injury. Importantly, circ_0114428 knockdown inhibited ROCK2 production by interacting with miR-574-5p.ConclusionCirc_0114428 knockdown protected against LPS-induced HPAEpiC injury through miR-574-5p/ROCK2 axis, providing a novel therapeutic target in sepsis-induced ALI.

Discovery of a novel ROCK2 ATP competitive inhibitor by DNA-encoded library selection

Neurodegeneration disorders, such as Alzheimer's disease (AD), have garnered significant attention due to their impact on individuals and society as a whole. Understanding the mechanisms behind these disorders and developing effective therapy strategies is of utmost importance. One potential therapeutic target that has emerged is Rho-associated coiled-coil containing protein kinase 2 (ROCK2), as its accumulation and activity have been closely linked to memory loss. In this report, we present the findings of a recent discovery involving a new molecule that has the ability to competitively inhibit ROCK2 activity. This molecule was identified through the utilization of a DNA-encoded library (DEL) screening platform. Following selection against ROCK2, an off-DNA compound was synthesized and examined to ascertain its inhibitory properties, selectivity, mechanism of action, and binding mode analysis. From the screening, compound CH-2 has demonstrated an IC50 value of 28 nM against ROCK2, while exhibiting a 5-fold selectivity over ROCK1. Further analysis through molecular docking has provided insights into the specific binding modes of this compound. Our findings suggest that DEL selection offers a rapid method for identifying new inhibitors. Among these, the CH-2 compound shows promise as a potential ROCK2 inhibitor and warrants further investigation.

Human Placental Mesenchymal Stem Cells-Exosomes Alleviate Endothelial Barrier Dysfunction via Cytoskeletal Remodeling through hsa-miR-148a-3p/ROCK1 Pathway.

Endothelial barrier disruption of human pulmonary vascular endothelial cells (HPVECs) is an important pathogenic factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Mesenchymal stem cells-exosome (MSCs-Exo) represents an ideal carrier for cell-free therapy. The therapeutic implication and underlying mechanism of human placental MSCs-Exo (HPMSCs-Exo) in ALI/ARDS need to be further explored. HPMSCs-Exo was extracted from HPMSCs and characterized. Then, the therapeutic effects of exosomes were evaluated in ALI mice and HPVECs. RNA-sequencing was applied to reveal the miRNA profile of HPMSCs-Exo and differentially expressed genes (DEGs) in HPMSCs-Exo-pretreated HPVECs. The targets of miRNAs were predicted by bioinformatics methods and correlated to DEGs. Finally, the role of hsa-miR-148a-3p/ROCK1 pathway in HPVECs has been further discussed. The results showed that HPMSCs-Exo could downregulate Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), upregulate the expression of zonula occludens-1 (ZO-1) and F-actin, promote HPVECs migration and tube formation, reduce cytoskeletal disorders and cell permeability, and thus improve ALI/ARDS. RNA-sequencing revealed the DEGs were mainly enriched in cell junction, angiogenesis, inflammation, and energy metabolism. HPMSCs-Exo contains multiple miRNAs which are associated with cytoskeletal function; the expression abundance of hsa-miR-148a-3p is the highest. Bioinformatic analysis identified ROCK1 as a target of hsa-miR-148a-3p. The overexpression of hsa-miR-148a-3p in HPMSCs-Exo promoted the migration and tube formation of HPVECs and reduced ROCK1 expression. However, the overexpression of ROCK1 on HPVECs reduced the therapeutic effect of HPMSCs-Exo. HPMSCs-Exo represents a protective regimen against endothelial barrier disruption of HPVECs in ALI/ARDS, and the hsa-miR-148a-3p/ROCK1 pathway plays an important role in this therapeutics implication.

Exploring the Role of ROCK Inhibition in Corneal Edema Through Crosstalk Between Epithelial and Endothelial Cells.

The maintenance of corneal transparency and normal vision is dependent on preservation of epithelial and endothelial cell layer homeostases. Different types of corneal injury can induce swelling and losses in transparency. Fuchs endothelial corneal dystrophy (FECD) is one type of injury that is commonly treated with rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors. While their clinical benefit is apparent, certain aspects of their mechanism of action require clarification. Specifically, although topical eye drops containing ROCK inhibitors have been employed to treat corneal endothelial dysfunction-associated corneal edema, it remains unclear whether interactions between both corneal epithelial and endothelial cell contribute to mitigating clinical signs that compromise normal vision. To address this question, we first review the intricate ROCK signaling pathways and their role in modulating a variety of functions that are related to the maintenance of corneal transparency and normal vision. We also review the results of ongoing clinical trials employing current FDA-approved ROCK inhibitors, highlighting the prominent role of Y-27632 in the treatment of a variety of ocular conditions, particularly FECD, and its promising results in reversing losses in normal vision through facilitating cell proliferation and suppressing apoptosis. This review shows that the ROCK inhibitor clinical benefit is affected by their interactions between the epithelium and the endothelium. This realization makes it likely that ROCK inhibitors will be approved for use in a clinical setting to treat FECD.

Selectivity matters: selective ROCK2 inhibitor ameliorates established liver fibrosis via targeting inflammation, fibrosis, and metabolism

The pathogenesis of hepatic fibrosis is driven by dysregulated metabolism precipitated by chronic inflammation. Rho-associated coiled-coil-containing protein kinases (ROCKs) have been implicated in these processes, however the ability of selective ROCK2 inhibition to target simultaneously profibrotic, pro-inflammatory and metabolic pathways remains undocumented. Here we show that therapeutic administration of GV101, a selective ROCK2 inhibitor with more than 1000-fold selectivity over ROCK1, attenuates established liver fibrosis induced by thioacetamide (TAA) in combination with high-fat diet in mice. GV101 treatment significantly reduces collagen levels in liver, associated with downregulation of pCofilin, pSTAT3, pAkt, while pSTAT5 and pAMPK levels are increased in tissues of treated mice. In vitro, GV101 inhibits profibrogenic markers expression in fibroblasts, adipogenesis in primary adipocytes and TLR-induced cytokine secretion in innate immune cells via targeting of Akt-mTOR-S6K signaling axis, further uncovering the ROCK2-specific complex mechanism of action and therapeutic potential of highly selective ROCK2 inhibitors in liver fibrosis.