ROCK Activity Research Articles

Intrarenal angiotensin II mediated ROCK1 activation promotes hypertension during chronic acidotic conditions

The newly reported model of experimental hypertension delineates the scope of chronic metabolic acidosis as a cause of hypertension. The observed mechanism to promote hypertension during the acidotic conditions are regulated by intrarenal angiotensin II (Ang II) and oxidative stress functioning synergistically. The present study investigates the role of Rho Kinase (ROCK1) in mediating intrarenal activation of Ang II and the renal cell oxidative state during the observed acidosis elicited hypertension. Chronic metabolic acidosis (CMA) was induced in male Sprague Dawley rats (100-150 g) by providing a weak acid solution of 0.28 M ammonium chloride (NH4CL) in tap water for 8 weeks. To confirm the rats were acidotic, blood pH was measured, while blood pressure was monitored weekly using tail-cuff. Rats were divided into five groups: Control (vehicle treated), CMA (acidotic), CMA+spironolactone (to evaluate aldosterone effect), CMA+captopril (to evaluate Ang II effect), and CMA+tempol (to evaluate free radical effect). Microdialysis probes were implanted to the rat’s renal compartments in each group to collect the interstitial fluid. ELISA was performed to quantifying the ROCK1 in all the dialysate samples. In the CMA rats, sustained elevation in the mean arterial pressure (MAP), associated with the significant decrease in blood pH was observed compared to the Control group over the 8 weeks. A significant decrease in MAP was observed in CMA rats treated with captopril and tempol, whereas spironolactone treatment caused no decrease in MAP as compared to the CMA group. The interstitial ROCK1 was significantly increased in the CMA group vs control but decreased significantly in the CMA+captopril vs CMA and CMA+tempol vs CMA groups respectively. This suggested the intrarenal ROCK1 is regulating the Ang II and ROS generation in the renal compartment. Overall, this supports our hypothesis that ROCK pathway and its interaction with intrarenal Ang II and oxidative stress plays a pivotal role in the pathogenesis of acidosis promoted hypertension. VCOM Departmental funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CircHECTD1 promoted MIRI-associated inflammation via inhibiting miR-138-5p and upregulating ROCK2.

Myocardial ischemia-reperfusion injury (MIRI) was often observed after surgeries, causing a lot of suffering to patients. Inflammation and apoptosis were critical determinants during MIRI. We conveyed experiments to reveal the regulatory functions of circHECTD1 in MIRI development. The Rat MIRI model was established and determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We analyzed cell apoptosis using TUNEL and flow cytometry. Proteins expression was evaluated by western blot. The RNA level was determined by qRT-PCR. Secreted inflammatory factors were analyzed by ELISA assay. To predict the interaction sequences on circHECTD1, miR-138-5p, and ROCK2, bioinformatics analysis was performed. Dual-luciferase assay was used to confirm these interaction sequences. CircHECTD1 and ROCK2 were upregulated in the rat MIRI model, while miR-138-5p was decreased. CircHECTD1 knockdown alleviated H/R-induced inflammation in H9c2 cells. Direct interaction and regulation of circHECTD1/miR-138-5p and miR-138-5p/ROCK2 were confirmed by dual-luciferase assay. CircHECTD1 promoted H/R-induced inflammation and cell apoptosis by inhibiting miR-138-5p. miR-138-5p alleviated H/R-induced inflammation, while ectopic ROCK2 antagonized such effect of miR-138-5p. Our research suggested that the circHECTD1-modulated miR-138-5p suppressing is responsible for ROCK2 activation during H/R-induced inflammatory response, providing a novel insight into MIRI-associated inflammation.

Fibroblast clearance of damaged tissue following laser ablation in engineered microtissues.

Although the mechanisms underlying wound healing are largely preserved across wound types, the method of injury can affect the healing process. For example, burn wounds are more likely to undergo hypertrophic scarring than are lacerations, perhaps due to the increased underlying damage that needs to be cleared. This tissue clearance is thought to be mainly managed by immune cells, but it is unclear if fibroblasts contribute to this process. Herein, we utilize a 3D in vitro model of stromal wound healing to investigate the differences between two modes of injury: laceration and laser ablation. We demonstrate that laser ablation creates a ring of damaged tissue around the wound that is cleared by fibroblasts prior to wound closure. This process is dependent on ROCK and dynamin activity, suggesting a phagocytic or endocytic process. Transmission electron microscopy of fibroblasts that have entered the wound area reveals large intracellular vacuoles containing fibrillar extracellular matrix. These results demonstrate a new model to study matrix clearance by fibroblasts in a 3D soft tissue. Because aberrant wound healing is thought to be caused by an imbalance between matrix degradation and production, this model, which captures both aspects, will be a valuable addition to the study of wound healing.

Orenburg Radial-Concentric Structure at Great Depth: Experimental Modeling under Fluid Dynamic Loads and Comparative Analysis of Sandstone Samples from Oil Fields in Western Siberia

The study of the deep structure of the Orenburg radial-concentric structure in the basement of the Russian Platform based on seismotectonic and geomorphological data has been carried out. Interpretation of temporal regional seismic profiles crossing the structure was performed, identifying the main deep faults and measuring their dip angles. It was revealed that the radial-concentric structure is a large flower structure. Its structure involves the main multidirectional deep faults dipping at angles of \(\perp \)60°–85° and limiting horst or reverse fault zones in the basement. The hierarchy of faults structure has been established. Oil and gas fields are confined to the main deep dislocations of the Orenburg structure and represent flower structures of minor sizes. An experimental modeling of the formation of “flower” structures was performed based on the analysis of the results of studies using data from the acoustic emission activity of rocks and under the action of fluid dynamic loads performed at the “UIK-AE” unit (Institute of Geophysics, Ural Branch of RAS, Russia) on sandstone core samples from reservoir rocks of oil fields in Western Siberia. The developed model showed that the formation of flower structures can occur without horizontal shear due to lateral compression and deep pressure. With the help of the seismo-acoustic emission method, characteristic noise was recorded during the formation of cracks in core samples, sound attenuation before splitting, and then its sharp explosive increase from a break in the continuity of the rock. Analysis of the core of wells drilled within the Orenburg radial-concentric structure revealed signs of hydrothermal fluid discharge in the sedimentary cover rocks, such as wavy (or flat) layering, bizarre textures and intervals of breccias, shell-shaped inclusions, stillotite seams, fluid textures. It is concluded that the genesis of the Orenburg radial-concentric structure is associated with the impact of deep heat flows, fluids, high pressure and has a commonality with the explosive ring structures of shields and platforms.

Surrounding rock activity law and support optimization of double flexible formwork wall gob-side entry retention in stratified mining coal seams

To ensure gas control in the layered mining of extra-thick coal seams in Baijigou Coal Mine—in addition to reducing the loss of coal resources and amount of roadway driving—this article proposes the gob-side entry retention technology of flexible formwork concrete, and retains the 010202 working face four-layered return air roadway as the 010203 working face gas drainage lane. By combining theory and field practice, a flexible formwork concrete wall + anchor cable joint support scheme is proposed to strengthen small coal pillars. Further, the design scheme for the aforementioned framework at the bottom of the layered mining region of an extra-thick coal seam is proposed. Then, field tests are carried out, and the surrounding rock activity law of the double flexible formwork wall entry retention in a layered mining coal seam is obtained. The results show that (i) 010202 the whole process of overburden collapse to stability at the stratified mining face is completed 60--200 m behind the face and (ii) the roadway deformation of the extra-thick coal seam in the Baijigou Coal mine can be divided into four stages, and the mining pressure of the gas drainage roadway basically presents a dynamic change of “pressurized stage – peak stage – stabilized pressure stage”. Based on this, three mechanical stages of roadway deformation are derived: (1) δz< Nmz<< Nwz; (2) Nmz < δz << Nwz; and (3) Nmz < δz< Nwz. Three repeated mining support optimization schemes are provided, one for each stage. Finally, the deformation and damage of the surrounding rock are effectively controlled, and the requirement that the gas drainage roadway is still intact under the influence of the 010203 working face mining is realized. This study has certain reference significance for gob-side entry retention in the bottom layer of stratified mining in extra-thick coal seams.

Actomyosin-mediated cellular tension promotes Yap nuclear translocation and myocardial proliferation through α5 integrin signaling.

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/β1 integrin and fibronectin matrix increase in response to actomyosin-mediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart.

Extraction of Low-Dimensional Structures of Noble and Rare Metals from Carbonaceous Ores Using Low-Temperature and Energy Impacts at Succeeding Stages of Raw Material Transformation

The possibility of extraction of metals from ores of different genesis, containing low-dimensional structures of rare and noble metals, increases their commodity value and, in a deficit for some types of metals, leads to the need to search and develop new nature-like technologies, which can be used to extract from ores of different genesis almost all valuable noble, rare earth and nonferrous metals regardless of their concentration. This article presents the results of studying the processes of comminution and flotation to extract low-dimensional structures of noble and rare metals from carbonaceous ores using low-temperature and energy impacts at successive stages of the ores’ transformation. With the use of modern mineralogical, physical and chemical methods of research of composition, structure and properties of ores, the initial samples, concentrates and tailings after enrichment were studied. During the study, it was established that the difficulty of extraction of strategic metals from carbonaceous hard-enriched ores consists in fine dissemination of valuable components in concentrator minerals, mutual penetration of ore mineralization into each other and into rock-forming minerals, and in proximity of physical, chemical and technological properties of minerals, which complicates selective extraction of valuable components in concentrates. Also, difficulties in enrichment are associated with high flotation activity of waste rock, which significantly reduces the quality of concentrates.

The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis.

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.

The Development of Mobile Geography Virtual Laboratory for Rock and Soil Practicum Studies

The concept of concrete lithosphere material has limitations in being verbally stimulated by the teacher, so students have difficulty understanding the material. During the learning process, lithosphere material is rarely carried out in practicum activities due to the lack of media that can involve students directly. This study aims to produce, measure feasibility, and determine the effectiveness of mobile geography virtual laboratory learning media. This research was included in R&D research by adopting a 4-D development model. The results obtained, namely the Mobile Geography Virtual Laboratory media, are feasible and effectively used in rock and soil practicum activities. The results of this research can be an innovative solution for geography learning, especially in adequately supporting the implementation of practicum activities so that students can become more active and independent.

Formation conditions of Ediacaran–Cambrian cherts in South China: Implications for marine redox conditions and paleoecology

The Ediacaran–Cambrian transition period was a critical time interval for the environmental fluctuations and organismal evolution in geological history, popularly known as the “Cambrian Explosion”. The diagenetic mechanism of the widely developed Liuchapo bedded cherts in South China remain unclear and controversial during this critical time interval. To address this, we examined silicon and oxygen isotopes, as well as major and trace elements in the Liuchapo cherts from three wells developed from slope to basin facies in South China. Results reveal that the bedded cherts from the three wells were affected by episodic hydrothermal activity, and the percentage of hydrothermal fluids is highest in the Xc1 well and lowest in the Xb1 well. The paleo-seawater temperature, calculated from chert oxygen isotope values, present a gradual increasing trend from shallow- to deep- areas in South China from 542 to 526 Ma. Redox proxies indicate that the water column of the Xa1 and Xc1 wells were dominated by suboxic condition, while those of the Xb1 well was mainly in oxic conditions. There should exist an oxygen minimum zone (OMZ) during the shelf and slope transition. A suitable temperature and oxic environment are conducive to biological reproduction, which is consistent with higher primary productivity and abundant acritarch fossils in the Xb1 well. Silicon isotope values range from −0.6 ‰ to 1.2 ‰ in the three wells. The diagenetic origin of the Xa1, Xb1, and Xc1 wells was the consequence of the mixture of hydrothermal fluid and seawater, biological activity, and direct hydrothermal precipitation, respectively. Due to the redox condition and seawater temperature variation, silica-secreting organisms should be relatively limited. The episodic hydrothermal fluid, biological activity, and replacement of precursor rock may jointly affect the formation of the Liuchapo bedded cherts during the Ediacaran–Cambrian transition.

β1 integrin monoclonal antibody treatment ameliorates cerebral cavernous malformations.

β1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active β1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of β1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting β1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.

The accumulation of miR-125b-5p is indispensable for efficient erythroblast enucleation

Erythroblast enucleation is a precisely regulated but not clearly understood process. Polycythemia shows pathological erythroblast enucleation, and we discovered a low miR-125b-5p level in terminal erythroblasts of patients with polycythemia vera (PV) compared to those of healthy controls. Exogenous upregulation of miR-125b-5p levels restored the enucleation rate to normal levels. Direct downregulation of miR-125b-5p in mouse erythroblasts simulated the enucleation issue found in patients with PV, and miR-125b-5p accumulation was found in enucleating erythroblasts, collectively suggesting the importance of miR-125b-5p accumulation for erythroblast enucleation. To elucidate the role of miR-125b-5p in enucleation, gain- and loss-of-function studies were performed. Overexpression of miR-125b-5p improved the enucleation of erythroleukemia cells and primary erythroblasts. Infused erythroblasts with higher levels of miR-125b-5p also exhibited accelerated enucleation. In contrast, miR-125b-5p inhibitors significantly suppressed erythrocyte enucleation. Intracellular imaging revealed that in addition to cytoskeletal assembly and nuclear condensation, miR-125b-5p overexpression resulted in mitochondrial reduction and depolarization. Real-time PCR, western blot analysis, luciferase reporter assays, small molecule inhibitor supplementation and gene rescue assays revealed that Bcl-2, as a direct target of miR-125b-5p, was one of the key mediators of miR-125b-5p during enucleation. Following suppression of Bcl-2, the activation of caspase-3 and subsequent activation of ROCK-1 resulted in cytoskeletal rearrangement and enucleation. In conclusion, this study is the first to reveal the pivotal role of miR-125b-5p in erythroblast enucleation.

Effect of tree species on the elemental composition of wood ashes and their fertilizer values on agricultural soils

AbstractWood ashes obtained from household heating and cooking are often applied to home gardens and arable fields by farmers. The effect of tree species and their locations on the elemental composition of wood ashes derived from domestic cooking and heating is unknown. The study aimed to discover the fertilizer values of wood ashes obtained from Betula pendula, Carpinus betulus, Fagus sylvatica, Larix decidua, Picea abies, Pinus sylvestris, Quercus robur, and Tilia cordata from two different localities, Hlinsko and Mšec, Czech Republic. The total element content in the ashes of dry wood samples (wood and bark) burnt at 460°C with a wood stove interfaced with a thermometer was determined using portable x‐ray spectrometry. The content (in g kg−1) of P (3.23–20.53), K (26.79–136.22), Ca (94.89–295.56), and S (2.97–11.75) in the ashes varies according to the tree species, locality, parent rock, and anthropogenic activities in the location of trees. Additionally, trace element contents ranged from 0.63–32.07 g Mn kg−1, 0.34–4.6 g Fe kg−1, 32.4–2062 mg Zn kg−1, 47.61–193.09 mg Cu kg−1, 3.99–21.53 mg Mo kg−1, and 1.50–6.62 mg Se kg−1. The pH of the ashes ranged from 8.71 to 11.54, suitable to alleviate soil acidity and a condition satisfying soil additive. A significant positive correlation between the contents of Cu, Sr, and Pb with the ashes of Picea abies, Larix decidua, Pinus sylvestris, and Betula pendula at Hlinsko is indicative of ancient anthropogenic activities input in the soil. The combustion of wood under home heating temperatures resulted in the concentration of most risk metal(loid)s, below permissible limits in agricultural soils. Application of wood ashes on arable fields requires considerable caution due to potentially toxic elements (Zn and Pb).

The Experimental Investigation on Mechanics and Damage Characteristics of the Aeolian Sand Paste-like Backfill Materials Based on Acoustic Emission.

With the wide application of the filling mining method, it is necessary to consider the influence of rock activity on the filling body, reflected in the laboratory, that is, the influence of loading rate. Therefore, to explore the response characteristics of loading rate on the mechanical and damage characteristics of aeolian sand paste filling body, DNS100 electronic universal testing machine and DS5-16B acoustic emission (AE) monitoring system were used to monitor the stress–strain changes and AE characteristic parameters changes of aeolian sand paste filling body during uniaxial compression, and the theoretical model of filling sample damage considering loading rate was established based on AE parameters. The experimental results show that: (1) With the increase in loading rate, the uniaxial compressive strength and elastic modulus of aeolian sand paste-like materials (ASPM) specimens are significantly improved. ASPM specimens have ductile failure characteristics, and the failure mode is unidirectional shear failure → tensile failure → bidirectional shear failure. (2) When the loading rate is low, the AE event points of ASPM specimens are more dispersed, and the large energy points are less. At high loading rates, the AE large energy events are more concentrated in the upper part, and the lower part is more distributed. (3) The proportion of the initial active stage is negatively correlated with the loading rate, and the proportion of the active stage is positively correlated with the loading rate. The total number of AE cumulative ringing decreases with the increase in loading rate. (4) Taking time as an intermediate variable, the coupling relationship between ASPM strain considering loading rate and the AE cumulative ringing count is constructed, and the damage and stress coupling model of ASPM specimen considering loading rate is further deduced. Comparing the theoretical model with the experimental results shows that the model can effectively reflect the damage evolution process of ASPM specimens during loading, especially at high loading rates. The research results have significant reference value for subsequent strength design of filling material, selection of laboratory loading rate and quality monitoring, and early warning of filling body in goaf.

Protective role of Decylubiquinone against secondary melanoma at lung in B16F10 induced mice by reducing E-cadherin expression and ameliorating ROCKII-Limk1/2-Cofiliin mediated metastasis

Melanoma is one of the most consequential skin cancer with a rising death incidences. Silent but belligerent nature of metastatic sprouting is the leading cause of melanoma related mortality. Invasion of metastatic cells and re-expression of E-Cadherin play the crucial role in the establishment of secondary tumor at distal sites. Thus, manipulation of tumor cell invasion in parallel to regulation of E-Cadherin expression can be considered as potential anti-metastatic strategy. Evidences suggested key role of reactive oxygen species associated ROCK activities in the modulation of metastatic invasion via F-actin stabilization. Here, we first-time report Decylubiquinone, a dietary Coenzyme Q10 analog, as an effective attenuator of pulmonary metastatic melanoma in C57BL/6 mice. Current study depicted detailed molecular interplay associated with Decylubiquinone mediated phosphorylation of ROCKII at Tyr722 along with reduced phosphorylation of ROCKII Ser1366 leading to suppression of Limk1/2-Cofilin-F-actin stabilization axis that finally restricted B16F10 melanoma cell invasion at metastatic site. Analysis further deciphered the role of HNF4α as its nuclear translocation modulated E-Cadherin expression, the effect of reactive oxygen species dependent ROCKII activity in secondarily colonized B16F10 melanoma cells at lungs. Thus unbosoming of related signal orchestra represented Decylubiquinone as a potential remedial agent against secondary lung melanoma.

Fasudil inhibits hepatic artery spasm by repressing the YAP/ERK/ ETA/ETB signaling pathway via inhibiting ROCK activation.

Objective: To explore the effect of Fasudil on HA spasm and its underlying mechanism.Methods: Rabbits were divided into Sham, Fasudil, and Model groups for experiments. Fasudil was injected into the left medial lobe of the rabbit liver using a 16G lumbar puncture needle through the laparotomic route. The spasm model was established by inserting the catheter sheath into the femoral arteries of rabbits, followed by celiac artery angiography and left HA catheterization with a micro-catheter. Next, the GSE60887 and GSE37924 datasets concerning Fasudil treatment were analyzed. Moreover, immunofluorescence staining was conducted for YAP1 and α-SMA. Finally, Western blotting was performed to examine the expressions of YAP1, ROCK, ERK1/2, ETA, and ETB.Results: Fasudil could relieve HA spasm. The Go and KEGG pathway analyses revealed that the MAPK signaling pathway and the Hippo signaling pathway were enriched in vasospasm. Besides, GSEA revealed that ROCK was functionally enriched in the MAPK and Hippo signaling pathways. Co-expression analysis revealed that MAPK1 was significantly correlated with YAP1 and MYC, and YAP1 was significantly correlated with ETA and ETB. It was manifested in the results of immunofluorescence staining that the YAP1-positive fluorescence area was significantly decreased after Fasudil treatment. Moreover, Western blotting results showed that Fasudil decreased the expressions of YAP1, RhoA, ROCK, ETA, ETB, and p-ERK1/2. In addition, in-vitro Western blotting revealed that Fasudil suppressed the YAP/ERK/ETA/ETB signaling pathway in the case of HA spasm by inhibiting ROCK activation.Conclusions: Fasudil ameliorates HA spasm through suppressing the YAP/ERK/ETA/ETB signaling pathway and the ROCK activation.

α-Actinin-4 recruits Shp2 into focal adhesions to potentiate ROCK2 activation in podocytes

Cell-matrix adhesions are mainly provided by integrin-mediated focal adhesions (FAs). We previously found that Shp2 is essential for FA maturation by promoting ROCK2 activation at FAs. In this study, we further delineated the role of α-actinin-4 in the FA recruitment and activation of Shp2. We used the conditional immortalized mouse podocytes to examine the role of α-actinin-4 in the regulation of Shp2 and ROCK2 signaling. After the induction of podocyte differentiation, Shp2 and ROCK2 were strongly activated, concomitant with the formation of matured FAs, stress fibers, and interdigitating intracellular junctions in a ROCK-dependent manner. Gene knockout of α-actinin-4 abolished the Shp2 activation and subsequently reduced matured FAs in podocytes. We also demonstrated that gene knockout of ROCK2 impaired the generation of contractility and interdigitating intercellular junctions. Our results reveal the role of α-actinin-4 in the recruitment of Shp2 at FAs to potentiate ROCK2 activation for the maintenance of cellular contractility and cytoskeletal architecture in the cultured podocytes.

Sex-based comparison of CD4+ T cell DNA methylation in lupus reveals proinflammatory epigenetic changes in men

Systemic lupus erythematosus (SLE) is more common in women than men, but the disease is more severe when it affects men. Lupus CD4+ T cells demonstrate dysregulated DNA methylation patterns. The purpose of this study was to investigate genome-wide CD4+ T cell differential DNA methylation between men (n = 12) and women (n = 10) with SLE. DNA methylation was evaluated using the Infinium MethylationEPIC array, and differences between male versus female SLE patients were calculated with probe-wise linear regressions with adjustment for age and disease activity. We identified 198 hypomethylated and 108 hypermethylated CpG sites in CD4+ T cells isolated from male compared to female SLE patients, annotated to 201 and 102 genes, respectively. A great proportion of these genes were related to apoptosis and immune functions. Among differentially methylated genes, CASP10, which is involved in the extrinsic apoptotic pathway, and multiple genes involved in T cell function and differentiation such as ELAVL1, UHRF1, and SMAD2, were hypomethylated in men compared to women with SLE. Importantly, network analysis of differentially methylated genes revealed a pattern consistent with increased activation of ROCK, PP2A, PI3K, and ERK1/ERK2 in men compared to women with SLE. These data provide epigenetic evidence suggesting activation of key T cell pathways in men compared to women with SLE and shed new light into possible mechanisms underlying increased SLE disease severity in men.

BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton.

The dynamics of the actin cytoskeleton and its connection to endothelial cell-cell junctions determine the barrier function of endothelial cells. The proper regulation of barrier opening/closing is necessary for the normal function of vessels, and its dysregulation can result in chronic and acute inflammation leading to edema formation. By using atomic force microscopy, we show here that thrombin-induced permeability of human umbilical vein endothelial cells, associated with actin stress fiber formation, stiffens the cell center. The depletion of the MEK/ERK kinase BRAF reduces thrombin-induced permeability prevents stress fiber formation and cell stiffening. The peripheral actin ring becomes stabilized by phosphorylated myosin light chain, while cofilin is excluded from the cell periphery. All these changes can be reverted by the inhibition of ROCK, but not of the MEK/ERK module. We propose that the balance between the binding of cofilin and myosin to F-actin in the cell periphery, which is regulated by the activity of ROCK, determines the local dynamics of actin reorganization, ultimately driving or preventing stress fiber formation.

Proximal-tubule molecular relay from early Protein diaphanous homolog 1 to late Rho-associated protein kinase 1 regulates kidney function in obesity-induced kidney damage

The small GTPase protein RhoA has two effectors, ROCK (Rho-associated protein kinase 1) and mDIA1 (protein diaphanous homolog 1), which cooperate reciprocally. However, temporal regulation of RhoA and its effectors in obesity-induced kidney damage remains unclear. Here, we investigated the role of RhoA activation in the proximal tubules at the early and late stages of obesity-induced kidney damage. In mice, a three-week high-fat-diet induced proximal tubule hypertrophy and damage without increased albuminuria, and RhoA/mDIA1 activation without ROCK activation. Conversely, a 12-week high-fat diet induced proximal tubule hypertrophy, proximal tubule damage, increased albuminuria, and RhoA/ROCK activation without mDIA1 elevation. Proximal tubule hypertrophy resulting from cell cycle arrest accompanied by downregulation of the multifunctional cyclin-dependent kinase inhibitor p27Kip1 was elicited by RhoA activation. Mice overexpressing proximal tubule-specific and dominant-negative RHOA display amelioration of high-fat diet-induced kidney hypertrophy, cell cycle abnormalities, inflammation, and renal impairment. In human proximal tubule cells, mechanical stretch mimicking hypertrophy activated ROCK, which triggered inflammation. In human kidney samples from normal individuals with a body mass index of about 25, proximal tubule cell size correlated with body mass index, proximal tubule cell damages, and mDIA1 expression. Thus, RhoA activation in proximal tubules is critical for the initiation and progression of obesity-induced kidney damage. Hence, the switch in the downstream RhoA effector in proximal tubule represents a transition from normal to pathogenic kidney adaptation and to body weight gain, leading to obesity-induced kidney damage.