Activation Of Rho-associated Kinase Research Articles

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.

A crucial stem cell plasticity regulation pathway: identification of key elements using the NCCIT human embryonic carcinoma cell line.

Upon removal of stemness factors, a small subpopulation of embryonic stem cells (ESCs) spontaneously extrudes the t-SNARE protein syntaxin-4, which upregulates the cell adhesion molecule P-cadherin and induces the onset of epithelial-mesenchymal transition (EMT)-like behaviors with loss of stemness in each cell. In this study, we identified a series of molecular elements responsible for this phenomenon using several small-molecule inhibitors and the human embryonic carcinoma cell line, NCCIT. We found that the syntaxin-4-triggered morphological changes and a decrease in stemness signatures were independently induced by the activation of Rho-associated kinase (ROCK) and the abrogation of PI3K/Akt signaling. We also found that the extracellular expression of syntaxin-4 inactivated focal adhesion kinase (FAK) in association with the augmented expression of P-cadherin, and comparable controls of either of these downstream elements of syntaxin-4 accelerated both ROCK-induced F-actin stress fiber formation and P13K/Akt-suppressed loss of stemness signatures. Cells expressing P-cadherin inactivated FAK but FAK inhibition did not affect P-cadherin expression, demonstrating a causal relationship between P-cadherin and FAK in the event of syntaxin-4 induction. These results reveal a novel signaling axis in stem cells and shed new light on the crucial elements for stem cell plasticity and the maintenance of stemness.

Ruscogenin ameliorates dasatinib-induced intestinal barrier dysfunction via ErbB4/YAP and ROCK/MLC pathways.

Dasatinib is effective in the treatment of chronic and acute myeloid leukemia, which could cause the side effect of gastrointestinal bleeding by overdose or longtime use. Ruscogenin (RUS) from the traditional Chinese medicine Ophiopogon japonicas could protect endothelial microvascular barrier function. In this study, the therapeutic effect and underlying mechanisms of RUS were investigated on intestinal barrier dysfunction induced by dasatinib. Male C57BL/6J mice were given three doses of dasatinib (70, 140, 210mg/kg, ig) and RUS (3, 10, 30μg/kg, ip) to explore the effect of dasatinib on intestinal barrier and the intervention of RUS. It was proved that dasatinib could reduce intestinal blood flow, inhibit phosphorylation of EGFR family member v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4 (ErbB4)/YES-associated protein (YAP) and activation of Rho-associated coiled coil-containing protein kinase (ROCK)/phosphorylation of (myosin light chain) MLC. RUS could significantly increase intestinal blood flow, improve intestinal injury, reduce Evans blue leakage and serum content of FITC-dextran 4kDa, and increase the expression of connexin (ZO-1, Occludin and VE-cadherin). Meanwhile, the in vitro effect of RUS (0.01, 0.1, 1μM) on the dysfunction of the endothelial barrier was observed in dasatinib (150nM)-pretreated HUVECs. The results showed that RUS suppressed dasatinib-induced the leakage of Evans blue, and degradation of F-actin and connexin. Furthermore, RUS could significantly increase the phosphorylation of ErbB4 at Tyr1284 site and YAP at Ser397 site, and inhibit ROCK expression and phosphorylation of MLC at Ser19 site in vivo and in vitro. In conclusion, the present research proved that RUS could suppress the side effects of dasatinib-induced intestinal barrier dysfunction by regulating ErbB4/YAP and ROCK/MLC pathways.

Regulation of enolase activation to promote neural protection and regeneration in spinal cord injury.

Spinal cord injury (SCI) is a debilitating condition characterized by damage to the spinal cord resulting in loss of function, mobility, and sensation with no U.S. Food and Drug Administration-approved cure. Enolase, a multifunctional glycolytic enzyme upregulated after SCI, promotes pro- and anti-inflammatory events and regulates functional recovery in SCI. Enolase is normally expressed in the cytosol, but the expression is upregulated at the cell surface following cellular injury, promoting glial cell activation and signal transduction pathway activation. SCI-induced microglia activation triggers pro-inflammatory mediators at the injury site, activating other immune cells and metabolic events, i.e., Rho-associated kinase, contributing to the neuroinflammation found in SCI. Enolase surface expression also activates cathepsin X, resulting in cleavage of the C-terminal end of neuron-specific enolase (NSE) and non-neuronal enolase (NNE). Fully functional enolase is necessary as NSE/NNE C-terminal proteins activate many neurotrophic processes, i.e., the plasminogen activation system, phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B, and mitogen-activated protein kinase/extracellular signal-regulated kinase. Studies here suggest an enolase inhibitor, ENOblock, attenuates the activation of Rho-associated kinase, which may decrease glial cell activation and promote functional recovery following SCI. Also, ENOblock inhibits cathepsin X, which may help prevent the cleavage of the neurotrophic C-terminal protein allowing full plasminogen activation and phosphatidylinositol-4,5-bisphosphate 3-kinase/mitogen-activated protein kinase activity. The combined NSE/cathepsin X inhibition may serve as a potential therapeutic strategy for preventing neuroinflammation/degeneration and promoting neural cell regeneration and recovery following SCI. The role of cell membrane-expressed enolase and associated metabolic events should be investigated to determine if the same strategies can be applied to other neurodegenerative diseases. Hence, this review discusses the importance of enolase activation and inhibition as a potential therapeutic target following SCI to promote neuronal survival and regeneration.

Abstract 14022: Involvement of Rho-Associated Coiled-Coil Containing Kinase (ROCK) in BCR-ABL1 Tyrosine Kinase Inhibitor Cardiovascular Toxicity

Introduction: Second and third generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) are associated with cardiovascular adverse events (CVAE) in patients with Philadelphia-chromosome positive (Ph+) leukemia. Hypothesis: We hypothesized that second and third generation BCR-ABL1 TKIs may cause CVAE through activation of Rho-associated protein kinase (ROCK). Methods: Peripheral blood mononuclear cells from 53 Ph+ patients on TKIs and 15 control patients without Ph+ leukemia were assessed for ROCK activity through capillary electrophoresis (median follow-up = 26 months [Q1-Q3, 5-37]). We also investigated the effects of TKIs and ROCK on endothelial dysfunction in vitro , which could contribute to CVAE. Results: Patients receiving second and third generation TKIs had 1.6-fold greater ROCK activity compared to both patients receiving imatinib and control patients. Elevated ROCK activity was associated with increased incidence of CVAE in Ph+ leukemia patients (Figure). In endothelial cells in vitro , we found that dasatinib and ponatinib treatment lead to changes in actin intensity and endothelial permeability, which can be reversed by pharmacologic inhibition of ROCK. Ponatinib leads to decreased cell proliferation, but this was not accompanied by senescence. Dasatinib and ponatinib treatment lead to phospho-inhibition of endothelial nitric oxide synthase (eNOS) and decreased nitric oxide production. ROCK inhibition reversed endothelial permeability and eNOS-related endothelial dysfunction. Imatinib and nilotinib induce phosphorylation of p190RhoGAP. Conclusions: Our findings suggest ROCK activity may be a prognostic indicator of CVAE in patients receiving BCR-ABL1 TKIs. ROCK inhibition may be a promising approach in reducing the incidence of CVAE associated with second and third generation BCR-ABL1 TKIs.

The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1-Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1-Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

Calcium‐dependent signaling via gap junctions drives RhoA/ROCK activation during collective cell migration

Collective cell migration (CCM) plays an important role in embryogenesis, vascular sprouting, and wound healing, but is also a significant driver of cancer metastasis. Ras homolog family member A (RhoA) facilitates CCM by modulating contraction of the actomyosin cytoskeleton. Given the precise spatiotemporal regulation of RhoA during CCM, we wanted to investigate the activity of downstream effector Rho‐associated kinase (ROCK) in collectively migrating fibroblasts. Using a new FRET‐based ROCK biosensor, we have observed calcium‐dependent activation of ROCK. We have previously demonstrated blunting of CCM in response to pharmacological inhibition of ROCK or depletion of intracellular calcium with 100 µM EGTA. To study the role of gap junction signaling in calcium‐dependent activation of ROCK during CCM, carbenoxolone, a gap junction blocker, was applied to collectively migrating cells following scratch wounding. Carbenoxolone treatment blunted CCM, as well as decreased ROCK activity and intracellular calcium levels at corresponding time points. Treatment with PQ7, a gap junction activator, caused modest increases in CCM. These results indicate that calcium signaling through gap junctions drives ROCK activity in the context of collective cell migration.

Chlorogenic acid improves the intestinal barrier by relieving endoplasmic reticulum stress and inhibiting ROCK/MLCK signaling pathways.

Phenolic acids play an active role in protecting the intestinal barrier, the structural integrity and function of which are crucial for host health. In the present study, we aimed to identify phenolic compounds that protect the intestine and explore the underlying mechanisms. We performed an imaging-based, quantitative, high-content screening (using Caco-2 and LS174T incubated with lipopolysaccharide/palmitic acid, respectively) to identify phenolic acids that could improve the mucosal barrier. We found that chlorogenic acid (CGA), 5-caffeoylquinic acid, protocatechuic acid, and caffeic acid alleviated intestinal barrier disruption. Furthermore, CGA increased transepithelial electrical resistance (TEER) and decreased paracellular permeability. Mechanistically, CGA inhibited the activation of myosin light chain kinase (MLCK) and Rho-associated kinase 1 (ROCK1) signals, thereby downregulating the expression of the downstream molecules phosphorylated myosin phosphatase target subunit 1 (p-MYPT1), MLCK, and phosphorylated myosin light chain (p-MLC), and upregulating the expression of tight junction proteins. In addition, CGA alleviated endoplasmic reticulum (ER) stress by inhibiting the expression levels of ER markers [glucose-regulated protein78 (GRP78) and C/EBP homologous protein (CHOP)] and the nuclear translocation of activating transcription factor 6 (ATF6), thereby promoting the expression of mucin [mucin 2 (Muc2), mucin 5AC (MUC5AC)] and secretory factor trefoil factor family 3 (TFF3) proteins. In summary, we identified four substances that can stabilise intestinal homeostasis. Of these, CGA protects the intestinal barrier by inhibiting ROCK/MLCK signalling pathways and relieving ER stress. These findings highlight the importance of rapidly screening potential active ingredients that benefit the intestinal barrier and provide a theoretical basis for enteral nutrition.

Acute glucose influx-induced mitochondrial hyperpolarization inactivates myosin phosphatase as a novel mechanism of vascular smooth muscle contraction

It is well-established that long-term exposure of the vasculature to metabolic disturbances leads to abnormal vascular tone, while the physiological regulation of vascular tone upon acute metabolic challenge remains unknown. Here, we found that acute glucose challenge induced transient increases in blood pressure and vascular constriction in humans and mice. Ex vivo study in isolated thoracic aortas from mice showed that glucose-induced vascular constriction is dependent on glucose oxidation in vascular smooth muscle cells. Specifically, mitochondrial membrane potential (ΔΨm), an essential component in glucose oxidation, was increased along with glucose influx and positively regulated vascular smooth muscle tone. Mechanistically, mitochondrial hyperpolarization inhibited the activity of myosin light chain phosphatase (MLCP) in a Ca2+-independent manner through activation of Rho-associated kinase, leading to cell contraction. However, ΔΨm regulated smooth muscle tone independently of the small G protein RhoA, a major regulator of Rho-associated kinase signaling. Furthermore, myosin phosphatase target subunit 1 (MYPT1) was found to be a key molecule in mediating MLCP activity regulated by ΔΨm. ΔΨm positively phosphorylated MYPT1, and either knockdown or knockout of MYPT1 abolished the effects of glucose in stimulating smooth muscle contraction. In addition, smooth muscle-specific Mypt1 knockout mice displayed blunted response to glucose challenge in blood pressure and vascular constriction and impaired clearance rate of circulating metabolites. These results suggested that glucose influx stimulates vascular smooth muscle contraction via mitochondrial hyperpolarization-inactivated myosin phosphatase, which represents a novel mechanism underlying vascular constriction and circulating metabolite clearance.

Yap activation in irradiated parotid salivary glands is regulated by ROCK activity.

Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients’ quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.

Uncovering Tumor-Stroma Inter-relationships Using MALDI Mass Spectrometry Imaging.

Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established breast cancer mouse model to investigate this inter-relationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify the proteomic changes occurring within tumors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic tool. Peptides were identified by liquid chromatography tandem mass spectrometry, and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely, collagen I, α-SMA, Rab14, and tubulin-β4. Rab14 and tubulin-β4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, correlates with cancer progression in two human breast cancer genomic data sets, and high expression of COL1A and ACTA2 (the gene encoding α-SMA) are associated with a low survival probability (COLIA, p = 0.00013; ACTA2, p = 0.0076) in estrogen receptor-negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated CAFs with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI-MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models.

RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization.

Background: In this study, we investigated the mechanism of Rho GTPases signaling on Ang II-mediated cell migration and dedifferentiation in human aortic vascular smooth muscle cells (HA-VSMCs) and an Ang II-infusion mouse model. Methods: Cells were pretreated with different inhibitors or Ang II. Cell migration was detected by Wound healing and Transwell assay. Mice were treated with Ad-RhoA-shRNA virus or Irbesartan or fasudil and then infused with Ang II. Results: Ang II treatment induced HA-VSMCs migration in a dose- and time-dependent manner and reduced the expression of VSMC contractile proteins. These effects were significantly suppressed by the inhibition of Ang II type 1 receptor (AT1 receptor), RhoA, and Rho-associated kinase (ROCK). Furthermore, Ang II treatment promoted the activation of RhoA and ROCK, which was reduced by AT1 receptor inhibition. Meanwhile, Ang II treatment induced F-actin polymerization, which was inhibited after ROCK inhibition. In mice, Ang II infusion increased VSMC migration into the neointima and reduced VSMC differentiation proteins levels, and these effects were shown to be dependent on AT1 receptor and RhoA/ROCK pathway. Conclusion: This study reveals a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation.

Overexpression of Na+/H+ exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment-mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.

Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes.

Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.

Irreversible Activation of Rho-activated Kinases Resulted from Evolution of Proteolytic Sites within Disordered Regions in Coiled-coil Domain.

Activation of Rho-associated protein kinase 1 (ROCK1) and myotonic dystrophy kinase-related CDC42-binding kinase alpha (MRCKα) by caspases during apoptosis in vertebrates represents a prototypical example of co-option of kinases by proteases. How caspases acquired the ability to control these proteins during evolution of vertebrates is still unknown. Here, we report a phylogenetic and molecular study on the acquisition of caspase-cleavage sites in the family of Rho-activated kinases (RaKs). We demonstrate that the acquisition of such sites has more frequently occurred in identifiable intrinsically disordered regions (IDRs) within or flanking the coiled-coil domain. Thanks to computational identification of IDRs in protein sequences of different organisms, we predicted and validated the independent evolution of two caspase-cleavage sites in ROCK of arthropods and the loss of one of the MRCKα caspase-cleavage sites in ray-finned fishes. In conclusion, we shed light on the propensity of RaKs to evolve novel proteolytic sites, causing kinase activation and uniform subcellular distribution.

β-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells.

Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4+ T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4+ T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4+ Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4+ Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.

Abstract 588: Corticotropin-releasing Hormone via Its Type2 Receptor Inhibits Insulin-stimulated NO Formation in HUVECs

Background: Stress may provoke endothelial dysfunction thereby inducing atherosclerotic cardiovascular diseases (ASCVD). Corticotropin-releasing hormone (CRH) is recognized as a key regulator of hypothalamo-pituitary-adrenal axis under stress, however, its role in endothelial function and ASCVD is not known well. Insulin can produce NO formation in endothelial cells through eNOS activation. We studied effect of CRH and urocortin 2 (UCN2) on insulin-stimulated NO formation in HUVECs. Methods and Results: HUVECs were incubated in serum-free media for 8 h prior to the experiment. Treatment of HUVECs with insulin (100 nM) enhanced NO production detected by 5 μM 4,5-diaminofluorescein-diacetate. Fluorescent and phase contrast images were obtained using an fluorescence microscope (Em max : 515 nm), using an exposure time based on control conditions, and mean fluorescence intensity was calculated with ImageJ. Insulin-stimulated NO production was markedly inhibited by either CRH or UCN2 (100 nM each), which was reversed mostly by 100 nM astressin 2B (CRH receptor type 2 (CRHR2) inhibitor) and partly by 100 nM NBI35965 (CRHR1 inhibitor). CRH induced Rho-associated kinase (ROCK) and p-IRS-1(Ser). Insulin (2nM) stimulated p-eNOS (Ser1177), which was inhibited by CRH. Conclusions: Taken together, CRH/UCN2 via mainly its type 2 receptor signaling can inhibit insulin-stimulated eNOS/NO pathway. This CRH’s inhibitory effect on insulin signaling is mediated at least partly by activation of ROCK/ p-IRS-1 (Ser).

Bradykinin increases blood pressure in endotoxemic rats

Despite the knowledge about the mechanisms triggered by bradykinin (BK), mainly its hypotensive effect, several aspects of biological effects of this peptide remain unclear, such as its release and action in endotoxemia. The main hypothesis of this study was that blood pressure responses to BK are impaired in experimental models of sepsis. Female Wistar rats (200–280 g) received intraperitoneal (i.p.) injections of sterile saline (1 ml/kg), or lipopolysaccharide (LPS; 1 mg/kg), and were anesthetized at 6, 24, 48, or 72 h after for direct measurement of the mean arterial pressure (MAP). The hypotensive effect of intravenous (i.v.) BK (6, 20 and 60 nmol/kg), and acetylcholine (ACh; 60 nmol/kg) were assessed before and after the treatment with prazosin (0.5 mg/kg i.v.), HOE 140 (20 nmol/kg s.c.), [Leu8]des‐Arg9‐BK (100 nmol/kg, i.v.), losartan (15 mg/kg, i.v.), Y‐27632 (0.1 mg/kg, i.v.), or indomethacin (10 mg/kg i.v.). We also evaluated the influence of endotoxemia on BK‐ and ACh‐induced vasodilation in the in vitro perfused mesenteric vascular bed obtained from both control and LPS 24 h groups. Moreover, the expression of bradykinin B2 and angiotensin II AT1 receptors, and the phosphorylated MYPT‐1 subunit of myosin phosphatase (pMYPT‐1) was detected by Western blotting in homogenates of resistance mesenteric arteries. In spite of the lack of changes in the MAP, and the hypotensive effects induced by BK and ACh remained unaltered in the LPS groups, LPS‐treated animals presented hematological evidence of systemic inflammation (i.e. leukocytosis, high levels of nitrate + nitrite), compared with the control group. Although endotoxemia have not changed the hypotensive effects induced by BK and ACh, BK‐induced hypotension in LPS treated animals (both 24 h and 48 h groups) was followed by an immediate and sustained pressor effect. For instance, the MAP increased by 9 ± 2, 18 ± 5, and 24 ± 3 mm Hg in the control group, and by 29 ± 4, 42 ± 3, and 56 ± 4 mm Hg in the LPS 24 h group, after administration of 6, 20, and 60 nmol/kg BK, respectively. This hypertensive response to BK was not reduced by the selective antagonist of the alpha‐1 adrenergic receptor prazosin, nor by the antagonist of the BK B1 receptor [Leu8]des‐Arg9‐BK, but it was vanished by HOE‐140, a selective BK B2 receptor antagonist, and losartan, an angiotensin II AT1 receptor antagonist. We also found that the hypertensive effect of angiotensin II (60 nmol/kg) was enhanced from 52 ± 5 mm Hg in control animals to 75 ± 3 mm Hg in the LPS 24 h group, an event that was fully prevented by HOE‐140. Although the cyclooxygenase inhibitor indomethacin has not had any influence on BK effects, the Rho‐associated kinase (ROCK) inhibitor Y‐27632 avoided the enhanced hypertensive effect of BK in LPS‐treated animals. Western blotting analysis revealed that B2 and AT1 receptors, and pMYPT‐1, were significantly increased in resistance arteries at 24 h after LPS, compared with samples from control animals. Notably, perfused mesenteric vascular bed obtained from the LPS 24 h group also displayed pressor responses to BK. These results disclose that in spite of the typical hypotension produced by BK in healthy animals, administration of BK in endotoxemic rats produces a biphasic effect, which is characterized by a hypotension followed by a sustained rise in blood pressure. The pressor effect of BK, induced by endotoxemia, appears to be mediated by both bradykinin B2 and angiotensin II AT1 receptors and is fully dependent on activation of ROCK.Support or Funding InformationCNPq (448738/2014‐7). This study was approved by CEUA/UFSC (PP00566).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation.

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

T1R3 homomeric sweet taste receptor regulates adipogenesis through Gαs-mediated microtubules disassembly and Rho activation in 3T3-L1 cells.

We previously reported that 3T3-L1 cells express a functional sweet taste receptor possibly as a T1R3 homomer that is coupled to Gs and negatively regulates adipogenesis by a Gαs-mediated but cAMP-independent mechanism. Here, we show that stimulation of this receptor with sucralose or saccharin induced disassembly of the microtubules in 3T3-L1 preadipocytes, which was attenuated by overexpression of the dominant-negative mutant of Gαs (Gαs-G226A). In contrast, overexpression of the constitutively active mutant of Gαs (Gαs-Q227L) as well as treatment with cholera toxin or isoproterenol but not with forskolin caused disassembly of the microtubules. Sweetener-induced microtubule disassembly was accompanied by activation of RhoA and Rho-associated kinase (ROCK). This was attenuated with by knockdown of GEF-H1, a microtubule-localized guanine nucleotide exchange factor for Rho GTPase. Furthermore, overexpression of the dominant-negative mutant of RhoA (RhoA-T19N) blocked sweetener-induced dephosphorylation of Akt and repression of PPARγ and C/EBPα in the early phase of adipogenic differentiation. These results suggest that the T1R3 homomeric sweet taste receptor negatively regulates adipogenesis through Gαs-mediated microtubule disassembly and consequent activation of the Rho/ROCK pathway.