Abnormal angiogenesis and increased vascular permeability of subchondral bone are key mechanisms related to osteoarthritis (OA). However, the precise mechanisms responsible for heightened vascular permeability in OA remain unclear. The present study used proteomics to identify protein expression in damaged subchondral bone compared with normal subchondral bone. The results suggest that Ras homolog family member A (RhoA) may be associated with the vascular permeability of subchondral bone and ferroptosis in OA. The results of analysis of clinical samples indicated a significant increase in expression of RhoA in the subchondral bone of OA. This were consistent with the proteomics findings. We found through western blotting, RT‑PCR, and immunofluorescence that RhoA significantly increased the permeability of endothelial cells (ECs) by inhibiting inter‑EC adhesion proteins (zona occludens‑1, connexin 43 and Vascular endothelial‑Cadherin) and actin filaments. Furthermore, RhoA induced ferroptosis core proteins (glutathione peroxidase 4,  solute carrier family 7 member 11 and acyl‑CoA synthase long‑chain family member 4, ACSL4) by influencing lipid peroxidation and mitochondrial function, leading to ferroptosis of ECs. This suggested an association between RhoA, ferroptosis and vascular permeability. Ferroptosis significantly increased permeability of ECs by inhibiting inter‑EC adhesion proteins. RhoA increased vascular permeability by inducing ferroptosis of ECs. In vivo, inhibition of RhoA and ferroptosis significantly mitigated progression of OA by alleviating cartilage degeneration and subchondral bone remodeling in mice with destabilization of the medial meniscus. In conclusion, the present findings indicated that RhoA enhanced vascular permeability in OA by inducing ferroptosis. This may serve as a novel strategy for the early prevention and treatment of OA.

Shortening of airway smooth muscle and bronchoconstriction are pathognomonic for asthma. Airway shortening occurs through calcium-dependent activation of myosin light chain kinase, and RhoA-dependent calcium sensitization, which inhibits myosin light chain phosphatase. The mechanism through which pro-contractile stimuli activate calcium sensitization is poorly understood. Our review of the literature suggests that pro-contractile G protein coupled receptors likely signal through G12/13 to activate RhoA and mediate calcium sensitization. This hypothesis is consistent with the effects of pro-contractile agonists on RhoA and Rho kinase activation, actin polymerization and myosin light chain phosphorylation. Recognizing the likely role of G12/13 signaling in the pathophysiology of asthma rationalizes the effects of pro-contractile stimuli on airway hyperresponsiveness, immune activation and airway remodeling, and suggests new approaches for asthma treatment.

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.

The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC \"inclusionopathy\" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.

Mechanical stimulation is the key physical factor in cell environment. Mechanotransduction acts as a fundamental regulator of cell behavior, regulating cell proliferation, differentiation, apoptosis, and exhibiting specific signature alterations during the pathological process. As research continues, the role of epigenetic science in mechanotransduction is attracting attention. However, the molecular mechanism of the synergistic effect between mechanotransduction and epigenetics in physiological and pathological processes has not been clarified. We focus on how histone modifications, as important components of epigenetics, are coordinated with multiple signaling pathways to control cell fate and disease progression. Specifically, we propose that histone modifications can form regulatory feedback loops with signaling pathways, that is, histone modifications can not only serve as downstream regulators of signaling pathways for target gene transcription but also provide feedback to regulate signaling pathways. Mechanotransduction and epigenetic changes could be potential markers and therapeutic targets in clinical practice.

BACKGROUND: Mechano-growth factor (MGF), which is a growth factor produced specifically in response to mechanical stimuli, with potential of tissue repair and regeneration. Our previous research has shown that MGF plays a crucial role in repair of damaged periodontal ligaments by promoting differentiation of periodontal ligament stem cells (PDLSCs). However, the molecular mechanism is not fully understood. This study aimed to investigated the regulatory effect of MGF on differentiation of PDLSCs and its molecular mechanism.
METHODS: Initially, we investigated how MGF impacts cell growth and differentiation, and the relationship with the activation of Fyn-p-YAPY357 and LATS1-p-YAPS127. Then, inhibitors were used to interfere Fyn phosphorylation to verify the role of Fyn-p-YAP Y357 signal after MGF stimulation; moreover, siRNA was used to downregulate YAP expression to clarify the function of YAP in PDLSCs proliferation and differentiation. Finally, after C3 was used to inhibit the RhoA expression, we explored the role of RhoA in the Fyn-p-YAP Y357 signaling pathway in PDLSCs proliferation and differentiation.
RESULTS: Our study revealed that MGF plays a regulatory role in promoting PDLSCs proliferation and fibrogenic differentiation by inducing Fyn-YAPY357 phosphorylation but not LATS1-YAP S127 phosphorylation. Moreover, the results indicated that Fyn could not activate YAP directly but rather activated YAP through RhoA in response to MGF stimulation.
CONCLUSIONS: The research findings indicated that the Fyn-RhoA-p-YAPY357 pathway is significant in facilitating the proliferation and fibrogenic differentiation of PDLSCs by MGF. Providing new ideas for the study of MGF in promoting periodontal regenerative repair.

The giant cytoskeletal protein obscurin contains multiple cell signaling domains that influence cell migration. Here, we follow each of these pathways, examine how these pathways modulate epithelial cell migration, and discuss the cross-talk between these pathways. Specifically, obscurin uses its PH domain to inhibit phosphoinositide-3-kinase (PI3K)-dependent migration and its RhoGEF domain to activate RhoA and slow cell migration. While obscurin\'s effect on the PI3K pathway agrees with the literature, obscurin\'s effect on the RhoA pathway runs counter to most other RhoA effectors, whose activation tends to lead to enhanced motility. Obscurin also phosphorylates cadherins, and this may also influence cell motility. When taken together, obscurin\'s ability to modulate three independent cell migration pathways is likely why obscurin knockout cells experience enhanced epithelial to mesenchymal transition, and why obscurin is a frequently mutated gene in several types of cancer.

Dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) cause diverse and largely distinct channelopathies, including inherited forms of neuromuscular disease, skeletal dysplasias, and arthropathy. Pathogenic TRPV4 mutations cause gain of ion channel function and toxicity that can be rescued by small molecule TRPV4 antagonists in cellular and animal models, suggesting that TRPV4 antagonism could be therapeutic for patients. Numerous variants in TRPV4 have been detected with targeted and whole exome/genome sequencing, but for the vast majority, their pathogenicity remains unclear. Here, we used a combination of clinical information and experimental structure-function analyses to evaluate 30 TRPV4 variants across various functional protein domains. We report clinical features of seven patients with TRPV4 variants of unknown significance and provide extensive functional characterization of these and an additional 17 variants, including structural position, ion channel function, subcellular localization, expression level, cytotoxicity, and protein-protein interactions. We find that gain-of-function mutations within the TRPV4 intracellular ankyrin repeat domain target charged amino acid residues important for RhoA interaction, whereas ankyrin repeat domain residues outside of the RhoA interface have normal or reduced ion channel activity. We further identify a cluster of gain-of-function variants within the intracellular intrinsically disordered region that may cause toxicity via altered interactions with membrane lipids. In contrast, assessed variants in the transmembrane domain and other regions of the intrinsically disordered region do not cause gain of function and are likely benign. Clinical features associated with gain of function and cytotoxicity include congenital onset of disease, vocal cord weakness, and motor predominant disease, whereas patients with likely benign variants often demonstrated late-onset and sensory-predominant disease. These results provide a framework for assessing additional TRPV4 variants with respect to likely pathogenicity, which will yield critical information to inform patient selection for future clinical trials for TRPV4 channelopathies.

Osteocytes are terminally differentiated cells derived from osteoblasts and are deeply embedded within the bone matrix. They play a critical role in bone remodeling by generating a lacuno-canalicular network (LCN) and controlling the transport of nutrients. Due to the absence of blood vessels within the bone matrix, it is widely believed that osteocytes develop in a hypoxic environment. However, the mechanisms of osteocytogenesis and the role of oxygen sensing in this process remain unclear. Hypoxia-inducible factors (HIFs) are major transcriptional factors involved in oxygen sensing. Previous studies have shown that accumulation of HIFs in osteoblasts leads to abnormal bone remodeling, potentially linked with the alterations of the LCN network. Specifically, HIF-1α is hypothesized to play a more significant role in regulating bone remodeling compared to HIF-2α. Therefore, we investigated the functions of HIF-1α in dendrite formation and the establishment of the LCN network during osteocytogenesis. Immunostaining and scanning electron microscopy revealed that the E11 protein aggregates to form a ring structure that defines the site for dendrite initiation. This process is followed by activation of the ERM/RhoA pathway and recruitment of matrix metalloproteinase 14 (MMP14) to facilitate extracellular matrix degradation, enabling dendrite elongation. However, both hypoxic treatment and overexpression of HIF-1α impair ring formation, resulting in reduced ERM/RhoA activity and decreased matrix degradation capability. These findings suggest that abnormal HIF-1α activity in local areas could contribute to impaired LCN network formation and abnormal bone remodeling observed in bone diseases such as osteopenia and aging.

BACKGROUND: Craniofacial 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.
METHODS: We 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.
RESULTS: The 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.
CONCLUSIONS: This 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.