Search Results (402)

K-Rev Interaction Trapped protein-1 (KRIT1) is a scaffold protein that forms functional protein complexes involved in physiologically important signaling networks. While it is primarily recognized for its association with Cerebral Cavernous Malformations (CCMs), KRIT1 may also play critical roles in tumor formation and the acquisition of malignant phenotypes, regulating cell adhesion, cytoskeletal dynamics, and angiogenesis. In this study, we investigated the role of KRIT1 in cancer cell migration and metastasis, with a focus on identifying novel interacting proteins and characterizing the intracellular signaling pathways activated upon its loss. By using a yeast two-hybrid screening, we identified Kinesin Family Member 1C (KIF1C), a protein involved in regulating podosome and invadopodium elongation, as a novel binding partner of KRIT1, and the interaction was confirmed in melanoma and epithelial cancer cells. In silico docking and interaction interface analyses supported the KRIT1–KIF1C interaction, providing structural insight into the binding mode as shown experimentally. We also found that SRC and focal adhesion kinase (FAK) phosphorylation, as well as Ras homolog family member A (RhoA) expression, represent additional pathways affected by the loss of KRIT1. This study confirms our earlier hypothesis that KRIT1 functions as a tumor suppressor and uncovers a compelling link between its loss and enhanced cancer aggressiveness. Full article

Background/Objectives: Rho-associated protein kinase inhibitors reduce intraocular pressure (IOP) by enhancing aqueous humor outflow through the trabecular meshwork–Schlemm’s canal pathway. However, it remains unclear whether the fixed-dose combination of ripasudil hydrochloride hydrate and brimonidine tartrate (GLAALPHA) enhances conventional aqueous outflow in vivo. Methods: This single-center randomized clinical trial included healthy adult volunteers who received GLAALPHA, a brimonidine tartrate–brinzolamide fixed-dose combination (Ailamide), or brimonidine tartrate monotherapy (Aiphagan) in a crossover sequence. The aqueous column width in the episcleral veins was assessed at baseline and at 2 h (primary outcome) and 8 h using hemoglobin video imaging. Results: Among 24 participants, analyses included 23 GLAALPHA-treated eyes, 21 Ailamide-treated eyes, and 22 Aiphagan-treated eyes. Two hours after instillation, the aqueous column width significantly increased from baseline only in the GLAALPHA group (p = 0.002). The percent increase in the aqueous column width at 2 h was significantly greater with GLAALPHA than with Ailamide (p = 0.039) and not significantly different between GLAALPHA and Aiphagan (p = 0.114). At 8 h, the aqueous column width did not differ from the baseline in any groups. Conclusions: In healthy adult eyes, GLAALPHA significantly increased the aqueous column width in the episcleral veins 2 h after instillation, indicating enhanced conventional aqueous outflow. These findings provide evidence that GLAALPHA promotes trabecular outflow beyond the effects of brimonidine tartrate-containing comparators and offer mechanistic insights into its action. Full article

Familial thoracic aortic aneurysm and dissection (FTAAD), caused by the pathogenic Myh11 K1256del variant, is characterized by impaired aortic contractility; however, how reduced contractility predisposes the aorta to dissection remains incompletely understood. In this study, we performed a data-driven trans-omic upstream analysis using Genome Enhancer to identify key regulatory mechanisms in aortas from Myh11 K1256del mice under baseline conditions, without exposure to exogenous pathological stimuli. Transcriptome analysis revealed enrichment of genes related to smooth muscle contraction and regulation of myosin light chain phosphatase activity. Upstream computational analysis of regulatory regions identified nuclear factor of activated T cells 1 and lymphoid enhancer-binding factor 1 as major transcription factors, and further highlighted Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) as a predicted central regulator of the dysregulated transcriptional network. Druggability analysis suggested ROCK1 and the JunB proto-oncogene AP-1 transcription factor subunit as potential therapeutic targets. Furthermore, it predicted 51 candidate therapeutants, including atorvastatin, GSK-269962A, and atovaquone. These findings indicate that even in the absence of overt pathological stimulation, aortic tissue carrying the Myh11 K1256del variant exhibits a transcriptional program centered on ROCK signaling, which may prime the aorta for maladaptive responses to additional stress and may enhance susceptibility to dissection. This computational analysis requires experimental validation, but may provide a hypothesis-generating framework for development of preventive pharmacological interventions against FTAAD. Full article

The purpose of our study was to assess if spinacetin (SPC), a flavonoid found in spinach, can alleviate the cyclophosphamide (CYP)-induced changes in cystometric and inflammatory parameters indicative of the development of hemorrhagic cystitis. The animal experiments were conducted in female Wistar rats. The cohort of 60 animals was grouped as follows: I—control, II—CYP group, III—SPC group, and IV—CYP + SPC group. The cystometry and biochemical analyses were performed after a fortnight of SPC administration. SPC was found to restore normal cystometric parameters in CYP-induced cystitis and, similarly, it normalized c-Fos expression changes in the central micturition regions. SPC further prevented a massive increase in the bladder wall thickness/permeability due to exposition to CYP administration. CYP instillation resulted in the elevation of biomarkers found in urine (brain-derived neurotrophic factor, BDNF, and nerve growth factor, NGF), and in the bladder detrusor muscle (Rho kinase and vesicular acetylcholine transporter, VAChT), which were successfully restored after administration of SPC. As for the biomarkers in the bladder urothelium, the CYP-induced increases in TNF-α, IL-1β, IL-6, calcitonin gene-related peptide (CGRP), malondialdehyde, 3-nitrotyrosine, insulin-like growth factor-binding protein 3 (IGFBP-3), occludin, organic cation transporter 3 (OCT-3), orosomucoid-1 (ORM1), pituitary adenylate cyclase receptor 1 (PAC1), synaptosomal-associated protein 23 (SNAP23), SNAP25, and synaptic vesicle glycoprotein (SV2A) levels were attenuated by SPC. Finally, CYP administration resulted in a decrease in the heparin-binding EGF-like growth factor (HB-EGF), hemopexin (HPX), T-H protein, and tight junction protein (Z01), and we noted the successful restoration of all these changes in concentrations after application of SPC. In summary, SPC robustly mitigated cyclophosphamide (CYP)-induced cystometric dysfunction and biochemical alterations characteristic of iatrogenic hemorrhagic cystitis. These findings position SPC as a compelling therapeutic candidate and warrant further translational investigation for the management of CYP-induced bladder injury. Full article

For decades, the pharmacological identity of Panax ginseng has been primarily attributed to triterpenoid saponins known as ginsenosides. However, accumulating evidence indicates that ginseng also contains a structurally distinct lipid–protein complex, termed gintonin, enriched in lysophosphatidic acid (LPA) species. Unlike ginsenosides, which predominantly exert modulatory effects on membrane dynamics and intracellular kinase pathways, gintonin directly activates LPA G protein-coupled receptors (GPCRs), thereby inducing rapid phospholipase C (PLC) activation and intracellular Ca²⁺ mobilization. Biochemical analyses have identified major LPA species within the gintonin fraction, including C16:0, C18:0, and C18:1, stabilized within a proteinaceous matrix that may influence receptor engagement kinetics. Pharmacological studies demonstrate that gintonin preferentially activates LPA₁ and LPA₃ receptor subtypes, triggering downstream signaling cascades involving MAPK, PI3K/Akt, and Rho pathways. These receptor-mediated effects occur on a rapid temporal scale, distinguishing gintonin from the slower transcriptional and kinase-modulating actions of ginsenosides. In this review, we synthesize current evidence regarding the chemical architecture, receptor pharmacology, and signaling dynamics of gintonin and propose a dual signaling framework in which steroid-like saponins and lipid GPCR ligands represent complementary molecular axes within P. ginseng. Recognition of this layered signaling organization refines the molecular understanding of ginseng biology and highlights gintonin as a unique plant-derived GPCR ligand system. Full article

Rho GTPases are a group of guanosine triphosphate (GTP)-binding proteins with a relative molecular weight of about 20–30 kD, and 22 different Rho GTPases have been identified in mammalian cells, among which RhoA, Rac1 and Cdc42 are the most well-studied. Rho-associated coiled coil forming protein kinase (ROCK) is the most well-researched downstream effector of Rho GTPases. The Rho/ROCK signaling pathway widely participates in the reorganization of the cytoskeleton through cascade phosphorylation/dephosphorylation reactions and modulates cellular biological behaviors including cell adhesion, migration and phenotypic transformation. Abnormal activation of the Rho/ROCK signaling pathway is closely associated with the occurrence and progression of acute kidney injury, diabetic nephropathy, hypertension-related nephropathy and chronic allograft nephropathy, which contributes to podocyte injury, renal tubular epithelial-to-mesenchymal transition (EMT), mesangial cell proliferation and inflammatory infiltration in the kidney. This review focuses on the research progress and regulatory mechanisms of the Rho/ROCK signaling pathway in the above four major kidney diseases and discusses the therapeutic potential of targeting this pathway for kidney disease treatment, aiming to provide new insights for elucidating the pathogenesis of kidney diseases and developing novel therapeutic strategies. Full article

Tubule-derived pro-fibrotic mediators are central for the development of kidney fibrosis. We previously showed that fibrotic stimuli activate and elevate GEF-H1 (ARHGEF2) in tubular cells, leading to RhoA-dependent fibrotic reprogramming. In search of new mechanisms of GEF-H1 regulation, here we used immunoprecipitation and proximity ligation assay to show interaction between GEF-H1 and Myotonic Dystrophy Kinase-related Cdc42-binding kinase (MRCK)α in tubular cells. MRCKα silencing elevated GEF-H1 activity, and induced GEF-H1-dependent RhoA activation, stress fibre formation and myosin light chain phosphorylation. MRCKα depletion also elevated phospho-cofilin levels in a RhoA-dependent manner. The fibrogenic cytokine TGFβ1 rapidly increased binding between GEF-H1 and MRCKα, while MRCKα silencing augmented TGFβ1-induced GEF-H1 activation, suggesting a negative feedback loop. An mRNA array detecting fibrogenic genes revealed increase in a subset of basal and TGFβ1-induced genes following MRCKα depletion. MRCKα silencing promoted nuclear translocation of the profibrotic transcriptional co-activator Myocardin-Related Transcription Factor (MRTF), and MRTF-A+B depletion prevented increase in ACTA2 (α-smooth muscle actin), a key marker of fibrotic reprogramming. Finally, total MRCKα mRNA was reduced in a murine kidney fibrosis model, and immunohistochemistry revealed a decrease in tubular MRCKα. Taken together, we identified MRCKα as a new suppressor of GEF-H1/RhoA/MRTF signaling. Reduced MRCKα expression in kidney fibrosis may promote tubular fibrotic gene expression. Full article

Background: To evaluate the effects of preoperative topical ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, on Schlemm’s canal (SC) morphology in patients with primary open-angle glaucoma (POAG). Methods: This study included 95 SC specimens obtained during trabeculectomy from 95 patients with POAG. Based on preoperative treatment, patients were divided into two groups: ripasudil (−) group (n = 68) receiving four topical medications [FP receptor agonist, β-blocker, carbonic anhydrase inhibitor (CAI), and α₂ agonist], and ripasudil (+) group (n = 27) receiving the same four medications plus ripasudil. SC morphology parameters were assessed in thrombomodulin (TBM)-stained sections, including length parameters [TBM-positive/negative and opened/closed SC lengths] and area parameters [TBM-positive/negative and opened SC areas]. Between-group comparisons were performed using unpaired t-tests, and multiple regression analysis was conducted to adjust for age, gender, preoperative intraocular pressure (IOP), and oral CAI use. Results: The ripasudil (+) group had significantly longer total SC length (TSC: 302.5 µm) than the ripasudil (−) group (273.0 µm, p = 0.023). Among area parameters, the ripasudil (+) group showed significantly larger opened SC area (OSC-A: 2689 µm² vs. 1881 µm², p = 0.008) and TBM-negative opened SC area (NOSC-A: 716 µm² vs. 305 µm², p = 0.001), whereas TBM-positive opened SC area (POSC-A) was not significantly different between groups (2001 µm² vs. 1575 µm², p = 0.096). After multivariate adjustment, ripasudil use remained significantly associated with longer TSC (p = 0.011) and larger OSC-A (p = 0.014) and NOSC-A (p = 0.001). Conclusions: Preoperative use of topical ripasudil was associated with preservation of SC lumen morphology, particularly in regions lacking SC endothelium. These findings provide a theoretical basis for therapeutic strategies employing ROCK inhibitors to maintain SC morphology and function. Full article

Background/Objectives: Accurate prognostic assessment remains crucial in metastatic renal cell carcinoma (mRCC), especially as treatment options have expanded beyond vascular endothelial growth factor (VEGF)-targeted therapies to include immune checkpoint inhibitors (ICIs) and ICI–TKI combinations. The widely used IMDC classification shows important limitations in the modern therapeutic era, highlighting the need for complementary prognostic tools. In this context, the Meet-URO and CANLPH scores—incorporating clinical, inflammatory, and nutritional markers—have emerged as promising alternatives. To evaluate and compare the prognostic performance of the Meet-URO and CANLPH scoring systems in a real-world mRCC cohort predominantly treated with first-line tyrosine kinase inhibitor (TKI) monotherapy due to limited access to ICI-based combinations. Methods: This retrospective single-center study included 112 patients with mRCC. The Meet-URO score was calculated for all patients, while the CANLPH score was assessed in 56 patients with complete laboratory data. CAR, NLR, and PHR were computed using baseline pre-treatment measurements. Overall survival (OS) and progression-free survival (PFS), the latter defined exclusively for first-line therapy, were estimated using the Kaplan–Meier method. Correlations between inflammatory markers and survival outcomes were analyzed using Spearman’s rho. Results: Meet-URO demonstrated clear prognostic stratification across all five categories, with the most favorable outcomes in score group 2 and progressively poorer OS and PFS in higher-risk groups. CANLPH also showed meaningful survival discrimination, with the highest inflammatory group (score 3) exhibiting markedly reduced OS and PFS. CAR was the strongest individual predictor of survival, while NLR and PHR showed weaker associations. Conclusions: Both Meet-URO and CANLPH provide strong, complementary prognostic information in mRCC, even in a cohort largely treated with TKI monotherapy. Their integration into routine risk assessment may enhance clinical decision-making, particularly in resource-limited settings. Full article

Background/Objectives: Retinitis pigmentosa is a degenerative retinal disease and a major cause of inherited blindness globally. The pro-survival kinase AKT is downregulated in degenerating photoreceptors in retinitis pigmentosa, and its activation has shown neuroprotective effects in retinitis pigmentosa and other neurodegenerative disorders. In this study, we evaluated the therapeutic potential of SC79, a pharmaceutical AKT activator, in two mouse models of retinitis pigmentosa, rd1.GFP and RhoP23H.GFP. Methods: SC79 was administered intravitreally at postnatal day 12 (P12) and analysis was conducted at P16. Results: SC79 at 10 µM was well tolerated in wildtype mice, with no reduction in retinal function or thickness. In rd1.GFP mice, SC79 partially preserved peripheral outer nuclear layer (ONL) thickness, improved rod photoreceptor-driven optomotor contrast sensitivity responses, and improved cone photoreceptor morphology. Immunohistochemistry of retinal sections indicated AKT-related protein expression changes in both sham and SC79-treated rd1.GFP retinas, with sham injections leading to decreases in this pathway and SC79 injections restoring this back to uninjected protein levels or higher, indicating the damage from intravitreal injections can induce AKT-related protein expression changes. In RhoP23H.GFP mice, changes to the visual response from the therapeutic effects of SC79 were not detectable. An increased dosage of SC79 at 100 µM was evaluated in wildtype mice and showed no major toxic effects, although it did not confer neuroprotective benefits in either disease model. Conclusions: These results demonstrate the potential therapeutic effect of AKT pathway modulation for preserving photoreceptors in recessive retinitis pigmentosa, with further optimisation of treatment delivery required. Full article

Objective: Airway hyperresponsiveness (AHR) is a hallmark feature of asthma; however, its precise molecular mechanisms remain incompletely defined. In this study, we investigated protein expression in airway smooth muscle that may contribute to AHR, using an experimental model of ovalbumin-induced allergic asthma. Methods: Guinea pigs were sensitized and challenged with ovalbumin. Airway responsiveness to histamine was assessed, and proteomic analysis of the tracheal tissue was conducted using electrophoresis followed by MALDI/TOF-TOF mass spectrometry. Specific protein bands corresponding to the myosin phosphatase target subunit 1 (MYPT1) were analyzed, and regulatory subunit of glycogen-targeted phosphatase 1 (RG1) was further evaluated through immunohistochemistry. Results: MYPT1, previously associated with AHR, was not detected in the proteomic analysis. Interestingly, an RG1 peptide was identified. Immunohistochemistry showed a differential expression pattern was observed for the RG1 and Rho-associated protein kinase 2 (ROCK2), both of which were significantly upregulated in airway smooth muscle and positively correlated with the degree of AHR. Moreover, a significant positive correlation was observed between RG1 and ROCK2 expression levels. MYPT1 and its phosphorylated forms (Thr⁶⁹⁶ and Thr⁸⁵⁰), along with ROCK1 immunostaining, did not differ from controls. Conclusions: These findings suggest that RG1, along with ROCK2, may play an important role in airway hyperresponsiveness characteristic of asthma. Full article

To better address the challenge of corneal ulcer healing, with already available standard agents, and those recently introduced, such as stable gastric pentadecapeptide BPC 157, we introduced a novel conceptual framework—the “triad” of corneal ulcer healing↔corneal neovascularization↔intraocular pressure—and extended it to avascular tissues such as tendon. Within this framework, cytoprotection serves as the unifying principle, underscoring that therapeutic effects are not isolated but interconnected. Preclinical studies with BPC 157 therapy, as a cytoprotection agent, illustrate this integration. BPC 157 rapidly normalizes elevated intraocular pressure in glaucomatous rats, preserves retinal integrity, restores pupil function, maintains corneal transparency during ulcer or abrasion healing, and counteracts both corneal neovascularization and dry eye. In parallel, its consistent efficacy in tendon injury models highlights a cytoprotective specificity across avascular tissues. The cornea’s “angiogenic privilege,” preserved during healing and tendon recovery together, provides strong proof of concept. Furthermore, mapping standard therapeutic agents used for corneal ulcers, neovascularization, or glaucoma onto this triad, and linking them with tendon healing, reveals both shared pathways and inconsistencies across existing drug classes. Analyzed were the ascorbate, fibronectin, hyaluronic acid, metalloproteinase inhibitors, EGF, FGF, NGF, insulin, and IGF-1 (corneal ulcer healing), the antiangiogenic agents (endostatin, PAI-1, PEDF, angiostatin, TSP-1, TSP-2, IFN-α), corticosteroids, NSAIDs, cyclosporine A, anti-VEGF drops (treatment of corneal neovascularization), and alpha 2-agonists, beta-blockers, carboanhydrase inhibitors, muscarinic agonists, Rho-kinase inhibitors, and prostaglandin analogs (glaucoma). Taken together, these findings advance cytoprotection as a unifying therapeutic paradigm, with BPC 157 emerging as its first exemplar, and encourage further translational research toward clinical application. Full article

Infertility affects about 17.5% of couples, with male factors accounting for approximately 50% of cases. Cytoskeletal remodeling is increasingly recognized as a critical component of male reproductive function, particularly in the regulation of testosterone synthesis by Leydig cells. However, the underlying molecular mechanisms remain poorly defined. Rho-associated coiled-coil-containing kinase (ROCK), a key cytoskeletal regulator, influences actin dynamics, impacting intracellular trafficking. In this study, we investigated the roles of ROCK1 and ROCK2 in Leydig cells using the TM3 cell model. Pharmacological inhibition of ROCK activity with Y-27632 impaired actin cytoskeleton organization, reduced the phosphorylation of LIMK, COFILIN, and MLC2, and disrupted the colocalization of F-actin with StAR and cholesterol, thereby decreasing testosterone production. Furthermore, RNA-seq revealed that hCG promotes transcription of steroidogenesis-related genes, while ROCK inhibition reverses this effect. Silencing of ROCK1 via siRNA mimicked the effects of ROCK-i, suppressing steroidogenic gene expression and testosterone synthesis. In contrast, ROCK2 knockdown enhanced testosterone secretion, promoted F-actin remodeling, and increased traffic of cholesterol targeting mitochondria. These opposing effects triggered distinct responses in the SCAP–SREBP2 axis, indicating a feedback mechanism regulating cholesterol homeostasis. Collectively, our findings uncover the isoform-specific roles of ROCK1 and ROCK2 in coordinating cytoskeletal dynamics and steroidogenic activity, providing new insights into the regulation of male reproductive endocrinology and identifying potential therapeutic targets for androgen deficiency and male infertility. Full article

Background: The impairment of neurite outgrowth is an early pathological hallmark underlying various neurodegenerative disorders. The promotion of neurite outgrowth was considered as a feasible strategy to treat neurodegenerative disorders. 9-Methylfascaplysin (9-MF), a marine-derived, bioactive compound, has exhibited multiple neuroprotective activities. Methods and Result: In this study, 9-MF at nanomolar concentrations promoted neurite outgrowth, upregulated the expression of growth-associated protein-43 (GAP-43), and increased the mitochondrial positive area with similar efficacy as retinoic acid in PC12 cells. 9-MF-associated differentiated expressed genes were enriched in mitochondria and synapse, forming a Rho-associated coiled-coil containing a protein kinase 2 (ROCK2)-centralized network. CMap analysis further identified positive connections between 9-MF-induced perturbation and perturbations caused by the inhibition of the ROCK2 pathway. Molecular docking analysis demonstrated a high binding affinity between 9-MF and ROCK2, indicating that 9-MF could inhibit ROCK2. Furthermore, 9-MF significantly reduced the phosphorylation of ROCK2 with a similar efficacy as fasudil, a ROCK2 inhibitor. Narciclasine, a known ROCK2 activator, almost completely abolished the effects of 9-MF on the induction of neurite outgrowth in PC12 cells. Conclusions: 9-MF effectively promoted neurite outgrowth possibly via the inhibition of ROCK2, providing supporting evidence that 9-MF might be developed as a novel neurological drug. Full article

The tumour microenvironment in pancreatic ductal adenocarcinoma (PDA) regulates vascular function and therapeutic response. P21-activated kinases (PAKs) regulate cytoskeletal dynamics and angiogenesis; however, their roles in vascular reprogramming and chemotherapy responses remain unclear. This study examined the effects of a PAK1 knockdown (PAK1KD) and a PAK4 knockout (PAK4KO) on vascular remodelling in PDA. Human PANC-1 wild-type (WT), PAK1KD, and PAK4KO cells were injected subcutaneously into the flanks of SCID mice followed gemcitabine treatment. The tumour growth, vascular density, pericyte coverage, adhesion molecules, and hypoxia were determined. A proteomics study was used to identify the molecular changes involved in the vascular pathways. PAK1KD suppressed tumour growth and angiogenesis, promoted vascular normalisation, reduced hypoxia, and increased stromal ICAM-1. PAK4KO inhibited tumour growth, enlarged vessels, enhanced angiogenesis, and reduced hypoxia. PAK4KO did not affect adhesion molecules in the absence of gemcitabine, but markedly upregulated ICAM-1 and VCAM-1 with gemcitabine. Additionally, PAK4KO promoted vascular mimicry (VM) with a compromised integrity in tumour-derived vessels, but enhanced the integrity in endothelial-derived vessels. The proteomics study confirmed the enrichment of molecules in fibronectin and the VEGF pathway in PAK4KO cancer cells, along with the upregulation of EphA2, RhoA, ROCK1, ROCK2, and components of the EPH-ephrin signalling pathway, linking to enhanced VM. Neither PAK1KD nor PAK4KO increased the gemcitabine efficacy. In conclusion, PAK1KD and PAK4KO suppressed tumour growth with distinct vascular effects, but failed to enhance the gemcitabine responses, suggesting that PAK targeting reprograms the PDA vasculature, but offers limited benefit in chemotherapy-resistant models. Full article