Rho-associated Coiled-coil Containing Protein Kinase Research Articles

S-08-3: ROCK2 SPECIFIC INHIBITION DECREASES TH17/TREG BALANCE AND REDUCES HYPERTENSION AND HYPERTENSION-ASSOCIATED END-ORGAN DAMAGE

Hypertension is characterized by immune activation and inflammatory end-organ damage, yet there are no therapies for hypertension that specifically target the immune system. We previously demonstrated a critical role for pro-inflammatory CD4+ T cells and their cytokines, interleukin 17A (IL-17A) and IL-21, in the full development of hypertension and hypertension-associated renal and vascular damage. Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been shown to function as a molecular switch upregulating Th17 and inhibiting anti-inflammatory regulatory T cell (Treg) differentiation in autoimmune diseases. We found that ROCK2 expression is upregulated in splenic and renal CD4+ T cells in a DOCA (deoxycorticosterone) salt model of hypertension. We hypothesized that hypertensive stimuli increase T cell ROCK2 activity, leading to increased Th17/Treg ratios, and that selective ROCK2 inhibition is a potential novel therapeutic target for hypertension-associated end-organ damage. We showed in vitro that KD025, an orally bioavailable ROCK2 inhibitor, inhibits Th17 proliferation and IL-17A/IL-21 production. We then tested ROCK2 inhibition in vivo where DOCA salt-induced hypertension (uninephrectomy + 100 mg DOCA pellet + 1% NaCl drinking water) was initiated in all mice allowing for hypertension to develop for 10 days, followed by treatment with KD025 (50 mg/kg daily, n = 11) or vehicle (n = 8) starting on day 11 through the remainder of the protocol. KD025 treatment significantly attenuated cardiac hypertrophy (8.7 ± 0.3 vs 7.6 ± 0.2 mg/grams body weight, p = 0.005) and left ventricular (LV) fibrosis (4.9 ± 1.7 vs 2.4 ± 1.3% LV area, p = 0.008). Flow cytometric analysis revealed that KD025 decreased F4/80+ infiltrating cardiac macrophages (24,000 ± 578 vs 18,918 ± 1,430 cells, p = 0.011) and decreased the Th17/Treg ratio (0.251 ± 0.041 vs 0.117 ± 0.009, p = 0.003). Furthermore, KD025 attenuated renal infiltration of CD3+ T cells (59.0 ± 5.6 vs 43.5 ± 3.8 cells/mg tissue, p = 0.019). Additional studies using the Angiotensin II (Ang II, 490ng/kg/min) model of hypertension demonstrated that KD025 treatment starting on Day 14 of Ang II infusion significantly reduced blood pressures from Day 14 (154 ± 5.5mmHg) to Day 28 of the study (141.9 ± 5.9mmHg, p = 0.043, n = 5) which was not observed in vehicle controls (Day 14 157.0 ± 5.4mmHg, Day 28 154.9 ± 3.7mmHg, n = 4). These data indicate that the ROCK2-specific inhibitor KD025 may be a novel therapeutic for the treatment of hypertension and the associated end-organ damage.

Deciphering the therapeutic role of Kigelia africana fruit in erectile dysfunction through metabolite profiling and molecular modelling

Kigelia africana herbal products are often used traditionally to treat erectile dysfunction and other sexual complaints, but the underlying mechanism is not yet understood. This study focused on profiling the bioactive constituents of Kigelia africana fruit (KAF) using spectroscopic techniques and providing the computational models of their interactions with phosphodiesterase (PDE5) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) targets associated with erectile dysfunction. Integrated FT-IR, HPLC-MS, and GC-MS analysis revealed 152 (C1–C152) KAF compounds with highly diverse functional groups and chemical properties. Molecular docking showed several hit compounds as potential inhibitors of PDE5 and ROCK2. Post docking MMGBSA, QSAR, predictive physicochemical analysis and AdmetSAR analysis revealed that most of the hit compounds are potential drug leads. Among these, hydroxydoxepin (C3) and ritodrine (C131) exhibited the strongest interactions with PDE5, while epigallocatechin 3-O-p-coumarate (C9) and chlorogenic acid (C91) had the strongest interaction with ROCK2. The thermodynamic parameters and trajectory clusters computed from the trajectories obtained from the 100 ns full atomistic molecular dynamic (MD) simulation indicated the structural stability and conformational flexibility of the selected complexes. The MD simulation-based MMPBSA calculation further revealed strong binding affinity and energy contribution by active site residues of PDE5 towards binding the selected KAF compounds. Various computational analyses employed revealed that the catalytic residues Gln817, Val782 and Phe786 of PDE5 exhibited high interaction potential and flexibility towards C3 and C131. Overall, hydroxydoxepin, ritodrine and other phytochemicals in Kigelia africana may account for the therapeutic role of this plant in erectile dysfunction.

Computational Analysis of miR-140 and miR-135 as Potential Targets to Develop Combinatorial Therapeutics for Degenerative Tendinopathy.

Degenerative tendinopathy, a condition causing movement restriction due to high pain, highly impacts productivity and quality of life. The healing process is a complex phenomenon and involves a series of intra-cellular and inter-cellular processes. Proliferation and differentiation of the tenocyte is a major and essential process to heal degenerative tendinopathy. The recent development in microRNA (miRNA)-mediated reprogramming of the cellular function through specific pathways opened door for the development of new regenerative therapeutics. Based on information about gene expression and regulation of tendon injury and healing, we attempted to evaluate the combinatorial effect of selected miRNAs for better healing of degenerative tendinopathy. The present study was designed to evaluate the combinatorial effect of two miRNAs (has-miR-140 and has-miR-135) in the healing process of the tendon. Publicly available information/data were retrieved from appropriate platforms such as PubMed. Only molecular data, directly associated with tendinopathies, including genes/proteins and miRNAs, were used in this study. The miRNAs involved in tendinopathy were analyzed by a Bioinformatics tools (e.g., TargetScan, miRDB, and the RNA22v2). Interactive involvement of the miRNAs with key proteins involved in tendinopathy was predicted by the Insilco approach. Based on information available in the public domain, tendon healing-associated miRNAs were predicted to explore their therapeutic potentials. Based on computation analysis, focusing on the potential regulatory effect on tendon healing, the miR-135 and miR-140 were selected for this study. These miRNAs were found as key players in tendon healing through Rho-associated coiled-coil containing protein kinase 1 (ROCK1), IGF-1/PI3K/Akt, PIN, and Wnt signaling pathways. It was also predicted that these miRNAs may reprogram the cells to induce proliferation and differentiation activity. Many miRNAs are likely to regulate genes important for the tendinopathy healing process, and the result of this study allows an approach for miRNA-mediated regeneration of the tenocyte for tendon healing. Based on computational analysis, the role of these miRNAs in different pathways was established, and the results provided insights into the combinatorial approach of miRNA-mediated cell reprogramming. In this study, the association between miRNAs and the disease was evaluated to correlate the tendinopathy genes and the relevant role of different miRNAs in their regulation. Through this study, it was established that the synergistic effect of more than one miRNA on directed reprogramming of the cell could be helpful in the regeneration of damaged tissue. It is anticipated that this study will be helpful for the design of miRNA cocktails for the orchestration of cellular reprogramming events.

CIRCKLHL2 KNOCKDOWN ALLEVIATES SEPSIS-INDUCED ACUTE LUNG INJURY BY REGULATING MIR-29B-3P MEDIATED ROCK1 EXPRESSION DOWN-REGULATION.

Background: Acute lung injury (ALI) induced by sepsis is distinguished by an inflammatory progression. Herein, we investigated the action of circular RNA kelch like family member 2 (circKlhl2) in sepsis-induced ALI. Methods: The animal or cell model of sepsis ALI was established by LPS stimulation. The contents of circKlhl2, microRNA-29b-3p (miR-29b-3p), rho-associated coiled-coil containing protein kinase 1 (ROCK1), CyclinD1, B-cell lymphoma-2 (Bcl-2), and cleaved-caspase 3 (C-caspase-3) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. Cell viability was assessed by cell counting kit 8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. The abundances of proinflammatory cytokines were detected by enzyme-linked immunosorbent assay. Besides, the targeted relationship between miR-29b-3p and circKlhl2 or ROCK1 was verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Results: Loss of circKlhl2 mitigated lung injury and proinflammatory cytokine expression in sepsis-ALI mice model and alleviated LPS-induced apoptosis and inflammatory response in microvascular endothelial cell (MPVECs) in vitro . The abundances of circKlhl2 and ROCK1 were boosted, while the miR-29b-3p level was diminished in the animal or cell model of sepsis-ALI. MiR-29b-3p inhibition abrogated circKlhl2 knockdown-mediated effects on MPVECs injury. Moreover, miR-29b-3p overexpression promoted cell proliferation and inhibited apoptosis and inflammation in LPS-treated MPVECs, while ROCK1 enhancement reversed these effects. Conclusion: CircKlhl2 expedited the sepsis-induced ALI by adjusting miR-29b-3p/ROCK1 axis.

Circ_0004104 participates in the regulation of ox-LDL-induced endothelial cells injury via miR-942-5p/ROCK2 axis

BackgroundCardiovascular disease was the most common disease among the elderly with high morbidity and mortality. Circ_0004104 was demonstrated to be involved in the regulation of atherosclerosis.MethodsQuantitative real-time polymerase chain reaction was employed to measure the expression of circ_0004104, miR-942-5p and Rho associated coiled-coil containing protein kinase 2 (ROCK2). Cell proliferation was tested by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis was measured by flow cytometry, and tube formation assay was used to detect the angiogenesis ability of cells. Western blot assay was performed to assess protein levels. Enzyme‑linked immunosorbent assay was used to detect the release of IL-1β and TNF-α. The relationship between miR-942-5p and circ_0004104 or ROCK2 was identified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay.ResultsOxidized low-density lipoprotein (ox-LDL) inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and promoted apoptosis in a dose-dependent manner. Circ_0004104 was increased in serum of atherosclerosis patients and ox-LDL-treated HUVECs, and silence of circ_0004104 promoted the proliferation of ox-LDL-exposed HUVECs and inhibited cell apoptosis. MiR-942-5p downregulation reversed si-circ_0004104-mediated influences in HUVECs upon ox-LDL exposure. ROCK2 was the target of miR-942-5p and circ_0004104 regulated the expression of ROCK2 through sponging miR-942-5p. ROCK2 abated the influences of miR-942-5p in ox-LDL-stimulated HUVECs. Circ_0004104 was increased in the exosomes derived from ox-LDL-exposed HUVECs, and the expression of circ_0004104 was promoted in HUVECs after stimulation with ox-LDL-treated HUVECs cells-derived exosomes.ConclusionCirc_0004104 downregulation receded ox-LDL-induced injury in HUVECs through miR-942-5p and ROCK2.

Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption

We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.

Belumosudil Impacts Immunosuppression Pharmacokinetics in Patients with Chronic Graft-Versus-Host Disease

*EG/MP Co-first authors Background: Belumosudil, a novel compound targeting the Rho-associated coiled-coil containing protein kinase 2 (ROCK2) was recently approved for the treatment of chronic graft-versus-host disease (cGVHD) in patients who failed at least two prior lines of systemic therapy (Cutler C, Blood 2021). Clinical evaluation of the pharmacokinetic (PK) effects of belumosudil (BEL) on other immunosuppressive therapies (IST) has not been performed. In vitro and pharmacokinetic modeling indicate BEL displays weak inhibition of CYP3A as well as inhibition of P-glycoprotein which are involved in the metabolism and excretion of several ISTs used concurrently for treatment of cGVHD such as tacrolimus (TAC), cyclosporine, and sirolimus (SIRO). Further evaluation of these potential interactions is crucial in optimizing initial immunosuppressive dosing with concurrent BEL use and to maintain therapeutic IST levels in cGVHD patients. The objective of this study was to determine the impact of BEL on plasma concentrations of tacrolimus and/or sirolimus in cGVHD patients. Methods: We retrospectivity evaluated 30 patients between February 2019 and July 2022 at Moffitt Cancer Center who received BEL while concurrently on TAC and/or SIRO for the treatment of cGVHD. Patients were included if they had baseline IST plasma concentration before and at least one IST level during BEL therapy. Patients receiving TAC commonly had a target range of 3-7 ng/mL while those receiving SIRO mostly had a target range of 5-10 ng/ml. IST Concentration to dose (C/D) ratios were analyzed prior to initiation of BEL and at three time points after initiation. Results: Median age was 63 years (range: 27-79); 86.7% received three or more lines of therapy and 60% had severe cGVHD per NIH consensus criteria (Jagasia MH, et al. Biol Blood Marrow Transplant. 2015). Over 50% of the patients had a Karnofsky Performance Status (KPS) less than 90% at time of BEL initiation. Additional baseline characteristics are described in Table 1. Patients taking SIRO (N = 26) or TAC (N = 12) concurrently with BEL had a statistically significant increase in mean C/D ratio between pre-BEL and all later C/D ratios while taking BEL (SIRO p<0.001, TAC p=0.014, Table 1). Upon co-administration of BEL, a higher proportion of patients on TAC and SIRO had supratherapeutic levels (25% TAC, 62% SIRO) at a median of 26 and 34.5 days, respectively (Figure 1). Most TAC levels became therapeutic after dose adjustments over evaluated time points (0%, 10% supratherapeutic), but SIRO levels were more likely to be supratherapeutic despite dose adjustment (33, 36% supratherapeutic) at later time points (Figure 1). The C/D ratio for both TAC and SIRO increased over the post-BEL assessment periods, with a significant difference between post-BEL 1 and 2 in the TAC group and a significant difference between post-BEL 1 and 3 in the SIRO group, indicating this drug interaction may require multiple dose adjustments (Figure 1). The increase in C/D ratio was significant in patients not taking a concurrent CYP3A inhibitor (N= 26, SIRO p<0.001, TAC p=0.001). The impact on the C/D ratio was less notable in patients taking a 3A4 inhibitor (N = 4) or BEL dosed daily combined with a proton pump inhibitor (N = 2), making it unclear if the potential effect of BEL is truly lessened in these patients or is reflective of small sample number. The interaction between multiple CYP3A interacting medications, such as concurrent TAC+SIRO+BEL (N = 8), is also unclear due to limited sample numbers. Eight patients discontinued BEL during the study period. After stopping BEL, 63% (N=5) required increased doses of TAC or SIRO and all 8 had a return of C/D ratios closer to pre-BEL. Conclusions: In this single center study, we found that BEL impacts IST pharmacokinetics with clinical significance requiring dose adjustments. Notably, 12/26 patients on SIRO had a level of >15 ng/mL during the study period, indicating significant risk if this interaction is unmonitored. No patient on TAC had a level >15 ng/mL, but patients in this study had lower TAC goal ranges. Based on these findings, empiric dose reductions of at least 25% for SIRO and TAC should be considered with co-administration of BEL with close monitoring of IST levels at baseline and periodically during initial months of concurrent therapy. Future studies are needed to confirm BEL impact on IST pharmacokinetics, particularly in patients taking strong CYP3A4 inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK/Rock Inhibitor TQ05105 for Glucocorticoid-Refractory or -Dependent Chronic Graft-Versus-Host Disease：Preliminary Analysis Results of a Phase 1b/2 Study

Introduction: Chronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic stem cell transplantation that is a leading cause of morbidity, mortality, and impaired quality of life. It is an immune-mediated inflammatory and fibrotic disorder. The Janus kinase (JAK) signaling pathways play an important role in immune-cell activation and tissue inflammation during cGVHD. The rho-associated coiled-coil-containing protein kinase-2 (ROCK2) signaling pathway regulates Th17/regulatory T cells balance and controls profibrotic pathways involving in progression of cGVHD. TQ05105 is a novel, oral dual JAK1/2 and ROCK1/2 inhibitor targeting inflammatory and fibrotic components of cGVHD. Selective JAK/ROCK inhibition with TQ05105 may offer a novel approach to the management of cGVHD. Methods: This phase1b/2, multicenter, open-label study of TQ05105 enrolled moderate or severe glucocorticoid-refractory or -dependent cGVHD patients who had received at least one prior lines of therapy (LOTs). This trial consist of 2 parts：a phase 1b dose escalation phase using a standard 3+3 design to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Phase 2 of this study is a expansion of TQ05105 at RP2D. The initial dose is 10mg BID, then reduced to 5mg BID in the event of unacceptable toxicity and increased to 15mg BID in the absence of dose limited toxicity. The primary endpoint of Phase 1b portion of the study was safety and RP2D. The key secondary end points were the best overall response (BOR, complete or partial response) defined per the 2014 NIH Consensus criteria, failure-free survival (FFS) at 6 month and improved score on the modified Lee Symptom Scale (LSS). Results: From May 2021, through May 2022, 22 patients were enrolled. Median age was 33 years (16-89), 63.6% male. The median time from cGVHD diagnosis to enrollment was 36 months. Patients enrolled in 10mg BID (n=7) and 15mg BID (n=15) had received a median of 4 prior lines of cGVHD therapy, respectively. 77.3% of patients had severe cGVHD. 57.1% of patients in 10mg BID and 53.3% in 15mg BID had involvement of ≥ 4 organs. The most frequently involved organs in 10mg BID and 15mg BID, respectively, are eyes (100%, 86.7%), mouth (57.1%, 93.3%), lungs (57.1%, 86.7%), skin (57.1%, 53.3%), and Joints and/or fascia (57.1%, 46.7%). The median treatment duration was 165 days (29-370). TQ05105 was well tolerated without dose-limiting toxicities in two doses. The RP2D was not determined and enrollment is ongoing. Commonly reported AEs (≥20%) were Epstein-Barr virus infections (36.4%), pulmonary infection (31.8%), upper respiratory tract infection (31.8%), leukopenia (27.3%), neutropenia (27.3%), hypercholesterolemia (27.3%). Grade 3 or higher AEs were reported in 10 patients and SAEs in 7 patients. No apparent increase in incidence of infection was observed. No cases of cytomegalovirus (CMV) infection or reactivation were reported with TQ05105. As of the data cutoff, there was no death and no relapse. Of the 22 patients, the BOR was 100% (7 of 7) in 10mg BID and 73.3% (11 of 15) in 15mg BID, and median duration of response was not reached. Responses were rapid, with 45% of responders achieving a response by the first assessment (at 4 weeks). Responses were achieved across key subgroups in 10mg BID and 15mg BID, with BOR of 100% (4 of 4) and 72.7% (8 of 11) in patients with ≥ 4 organs involved, 100% (6 of 6) and 73.3% (11 of 15) in patients who had received ≥ 2 prior systemic LOTs, 100% (4 of 4) and 76.9% (10 of 13) in patients who were refractory to their last LOT before enrollment，100% (5 of 5) and 66.7%(8 of 12) in patients with severe cGVHD. The FFS rate was 88.9% (95%CI, 43.3%-98.4%) at 6 months. During TQ05105 treatment, 85.7% (6 of 7) in 10mg BID and 53.3% (8 of 15) in 15mg BID reduced corticosteroids (CS) dose. 8/22 (36.4%) patients discontinued CS. 57.1% (4 of 7) and 26.7% (4 of 15) of patients in 10mg BID and 15mg BID, respectively, achieved an improvement (≥ 7 points reduction) in the LSS Score, including both responders and non-responders. Conclusion: TQ05105 is well-tolerated for cGVHD and yielding high rates of clinical response, improving quality-of-life, reducing corticosteroids dose. The data indicated that TQ05105 may be proved to be a potential, effective therapy for patients with glucocorticoid-refractory or -dependent cGVHD. (Funded by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. ClinicalTrials.gov number, NCT04944043) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ROCK1/MLC2 inhibition induces decay of viral mRNA in BPXV infected cells

Rho-associated coiled-coil containing protein kinase 1 (ROCK1) intracellular cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. We observed the activation of ROCK1/myosin light chain (MLC2) signaling pathway in buffalopox virus (BPXV) infected Vero cells. ROCK1 depletion by siRNA and specific small molecule chemical inhibitors (Thiazovivin and Y27632) resulted in a reduced BPXV replication, as evidenced by reductions in viral mRNA/protein synthesis, genome copy numbers and progeny virus particles. Further, we demonstrated that ROCK1 inhibition promotes deadenylation of viral mRNA (mRNA decay), mediated via inhibiting interaction with PABP [(poly(A)-binding protein] and enhancing the expression of CCR4-NOT (a multi-protein complex that plays an important role in deadenylation of mRNA). In addition, ROCK1/MLC2 mediated cell contraction, and perinuclear accumulation of p-MLC2 was shown to positively correlate with viral mRNA/protein synthesis. Finally, it was demonstrated that the long-term sequential passage (P = 50) of BPXV in the presence of Thiazovivin does not select for any drug-resistant virus variants. In conclusion, ROCK1/MLC2 cell signaling pathway facilitates BPXV replication by preventing viral mRNA decay and that the inhibitors targeting this pathway may have novel therapeutic effects against buffalopox.

CircPalm2 knockdown alleviates LPS-evoked pulmonary microvascular endothelial cell apoptosis and inflammation via miR-450b-5p/ROCK1 axis

BackgroundEmerging evidence has revealed that circular RNAs (circRNAs) have roles in regulating the complex pathologies of sepsis-induced acute lung injury (sepsis-ALI). Herein, this work aimed to investigate the potential role and mechanism of circPalm2 in the process of sepsis-ALI. MethodsPrimary mice pulmonary microvascular endothelial cells (MPVECs) in functional group were exposed to lipopolysaccharide (LPS). Levels of genes and proteins were measured by qRT-PCR and western blotting. Functional experiments were conducted using In vitro functional experiments were conducted using cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry and ELISA analysis. The binding between miR-450b-5p and circPalm2 or ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1) was validated using dual-luciferase reporter, RNA immunoprecipitation and pull-down assays. ResultsLPS treatment caused the increase of circPalm2 and ROCK1, as well as the decrease of miR-450b-5p in MPVECs. Knockdown of circPalm2 attenuated LPS-induced proliferation arrest, apoptosis, and production of proinflammatory cytokine IL-6, IL-β and TNF-α in MPVECs. Mechanistically, circPalm2 sequestered miR-450b-5p to up-regulate ROCK1 expression, revealing the circPalm2/miR-450b-5p/ROCK1 feedback loop. Moreover, the protective functions mediated by circPalm2 silencing on MPVECs under LPS exposure were abolished by miR-129-5p inhibition or ROCK1 overexpression. ConclusionCircPalm2 knockdown can alleviate LPS-evoked MPVEC apoptosis and inflammation via miR-450b-5p/ROCK1 axis, suggesting the potential involvement of this ceRNA network in sepsis-ALI and a broader approach for the therapy of sepsis-ALI.

Trophectoderm formation: regulation of morphogenesis and gene expressions by RHO, ROCK, cell polarity, and HIPPO signaling.

Trophectoderm is the first tissue to differentiate in the early mammalian embryo and is essential for hatching, implantation, and placentation. This review article discusses the roles of Ras homolog family members (RHO) and RHO-associated coiled-coil containing protein kinases (ROCK) in the molecular and cellular regulation of trophectoderm formation. The trophectoderm (TE) is the first tissue to differentiate during the preimplantation development of placental mammals. It constitutes the outer epithelial layer of the blastocyst and is responsible for hatching, uterine attachment, and placentation. Thus, its formation is the key initial step that enables the viviparity of mammals. Here, we first describe the general features of TE formation at the morphological and molecular levels. Prospective TE cells form an epithelial layer enclosing an expanding fluid-filled cavity by establishing the apical-basal cell polarity, intercellular junctions, microlumen, and osmotic gradient. A unique set of genes is expressed in TE that encode the transcription factors essential for the development of trophoblasts of the placenta upon implantation. TE-specific gene expressions are driven by the inhibition of HIPPO signaling, which is dependent on the prior establishment of the apical-basal polarity. We then discuss the specific roles of RHO and ROCK as essential regulators of TE formation. RHO and ROCK modulate the actomyosin cytoskeleton, apical-basal polarity, intercellular junctions, and HIPPO signaling, thereby orchestrating the epithelialization and gene expressions in TE. Knowledge of the molecular mechanisms underlying TE formation is crucial for assisted reproductive technologies in human and farm animals, as it provides foundation to help improve procedures for embryo handling and selection to achieve better reproductive outcomes.

ROCK2 inhibition: A futuristic approach for the management of Alzheimer’s disease

Neurons depend on mitochondrial functions for membrane excitability, neurotransmission, and plasticity. Mitochondrial dynamics are important for neural cell maintenance. To maintain mitochondrial homeostasis, lysosomes remove dysfunctional mitochondria through mitophagy. Mitophagy promotes mitochondrial turnover and prevents the accumulation of dysfunctional mitochondria. In many neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), mitophagy is disrupted in neurons. Mitophagy is regulated by several proteins; recently, Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been suggested to negatively regulate the Parkin-dependent mitophagy pathway. Thus, ROCK2 inhibition may be a promising therapy for NDDs. This review summarizes the mitophagy pathway, the role of ROCK2 in Parkin-dependent mitophagy regulation, and mitophagy impairment in the pathology of AD. We further discuss different ROCK inhibitors (synthetic drugs, natural compounds, and gene therapy-based approaches) and examine their effects on triggering neuronal growth and neuroprotection in AD and other NDDs. This comprehensive overview of the role of ROCK in mitophagy inhibition provides a possible explanation for the significance of ROCK inhibitors in the therapeutic management of AD and other NDDs.

Implication of non-coding RNA-mediated ROCK1 regulation in various diseases.

Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) is a protein serine/threonine kinase which is activated upon binding with the GTP-bound form of Rho. This protein can modulate actin-myosin contraction and stability. Moreover, it has a crucial role in the regulation of cell polarity. Therefore, it participates in modulation of cell morphology, regulation of expression of genes, cell proliferation and differentiation, apoptotic processes as well as oncogenic processes. Recent studies have highlighted interactions between ROCK1 and several non-coding RNAs, namely microRNAs, circular RNAs and long non-coding RNAs. Such interactions can be a target of medications. In fact, it seems that the interactions are implicated in therapeutic response to several medications. In the current review, we aimed to explain the impact of these interactions in the pathoetiology of cancers as well as non-malignant disorders.

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

Chrysin-Induced G Protein-Coupled Estrogen Receptor Activation Suppresses Pancreatic Cancer.

Pancreatic cancer (PC) has a high mortality rate due to its poor prognosis and the possibility of surgical resection in patients with the disease. Importantly, adjuvant chemotherapy is necessary to improve PC prognosis. Chrysin, a natural product with anti-inflammatory, antioxidant, and anticancer properties, has been studied for several years. Our previous study demonstrated that chrysin induced G protein-coupled estrogen receptor (GPER) expression and regulated its activity in breast cancer. Herein, we investigated whether chrysin-induced GPER activation suppresses PC progression in MIA PaCa-2 cells and a xenograft model. To determine its mechanism of action, cytotoxicity and clonogenic assays, a FACS analysis, and Western blotting were performed. Furthermore, the delay in tumor growth was evaluated in the MIA PaCa-2-derived xenograft model. Tumor tissues were investigated by Western blotting, immunohistochemistry, and a proteomic analysis. Chrysin caused cell cycle arrest and significantly decreased cell viability. Following co-treatment with chrysin and 17β-estradiol, the inhibitory effect of chrysin on cell proliferation was enhanced. In the xenograft model, chrysin and G1 (a GPER agonist) significantly delayed tumor growth and reduced both Ki-67 (a proliferation marker) and c-Myc expressions in tumor tissues. The proteomic analysis of tumor tissues identified that rho-associated coiled-coil containing protein kinase 1 (ROCK1), transgelin 2 (TAGLN2), and FCH and Mu domain containing endocytic adaptor 2 (FCHO2) levels were significantly reduced in chrysin-treated tumor tissues. High ROCK1, TAGLN2, and FCHO2 expressions were indicative of low overall PC survival as found using the Kaplan–Meier plotter. In conclusion, our results suggest that chrysin suppresses PC progression through the activation of GPER and reductions in ROCK1, TAGLN2, and FCHO2 expressions.

Tas2R signaling enhances mouse neutrophil migration via a ROCK-dependent pathway.

Type-2 bitter taste receptors (Tas2Rs) are a large family of G protein-coupled receptors that are expressed in the oral cavity and serve to detect substances with bitter tastes in foods and medicines. Recent evidence suggests that Tas2Rs are also expressed extraorally, including in immune cells. However, the role of Tas2Rs in immune cells remains controversial. Here, we demonstrate that Tas2R126, Tas2R135, and Tas2R143 are expressed in mouse neutrophils, but not in other immune cells such as macrophages or T and B lymphocytes. Treatment of bone marrow-derived neutrophils from wild-type mice with the Tas2R126/143 agonists arbutin and d-salicin led to enhanced C-X-C motif chemokine ligand 2 (CXCL2)-stimulated migration in vitro, but this response was not observed in neutrophils from Tas2r126/135/143-deficient mice. Enhancement of CXCL2-stimulated migration by Tas2R agonists was accompanied by increased phosphorylation of myosin light chain 2 (MLC2) and was blocked by pretreatment of neutrophils with inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK), but not by inhibitors of the small GTPase RhoA. Taken together, these results demonstrate that mouse neutrophils express functional Tas2R126/143 and suggest a role for Tas2R126/143-ROCK-MLC2-dependent signaling in the regulation of neutrophil migration.

Pleiotropic Benefits of Statins in Cardiovascular Diseases.

In 1976, Japanese microbiologist Akira Endo discovered the first statin as a product of the fungus Penicillium citrinum that inhibited the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Their primary mode of action is to lower the blood cholesterol by decreasing hepatic cholesterol production, which upregulates hepatic low-density lipoprotein (LDL) receptors and increases LDL-cholesterol clearance. In addition to cholesterol lowering, statins inhibit other downstream products of the mevalonate pathway, causing the so-called pleiotropic effects. As a result of their pleiotropic effects statins modulate virtually all known processes of atherosclerosis and have beneficial effects outside the cardiovascular system Statins inhibit the post-translational prenylation of small GTP-binding proteins such as Rho, Rac, as well as their downstream effectors such as Rho kinase and nicotinamide adenine dinucleotide phosphate oxidases since they suppress the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway altering the expression of endothelial nitric oxide synthase, the stability of atherosclerotic plaques, production of proinflammatory cytokines, reactive oxygen species, platelet reactivity, development of cardiac hypertrophy and fibrosis in cell culture and animal experiments. Inhibition of Rho and Rho-associated coiled-coil containing protein kinase (ROCK), has emerged as the principle mechanisms underlying the pleiotropic effects of statins. However, the relative contributions of statin pleiotropy to clinical outcomes are debatable and difficult to measure because the amount of isoprenoid inhibition by statins corresponds to some extent with the amount of LDL-cholesterol decrease. This article examines some of the existing molecular explanations underlying statin pleiotropy and discusses if they have clinical relevance in cardiovascular diseases.

Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress

Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.

Sirt6 deficiency contributes to mitochondrial fission and oxidative damage in podocytes via ROCK1-Drp1 signalling pathway.

ObjectivesIncreasing evidence suggests that mitochondrial dysfunction is the key driver of angiotensin II (Ang II)‐induced kidney injury. This study was designed to investigate whether Sirtuin 6 (Sirt6) could affect Ang II‐induced mitochondrial damage and the potential mechanisms.Materials and MethodsPodocyte‐specific Sirt6 knockout mice were infused with Ang II and cultured podocytes were stimulated with Ang II to evaluate the effects of Sirt6 on mitochondrial structure and function in podocytes. Immunofluorescence staining was used to detect protein expression and mitochondrial morphology in vitro. Electron microscopy was used to assess mitochondrial morphology in mice. Western blotting was used to quantify protein expression.ResultsMitochondrial fission and decreased Sirt6 expression were observed in podocytes from Ang II‐infused mice. In Sirt6‐deficient mice, Ang II infusion induced increased apoptosis and mitochondrial fragmentation in podocytes than that in Ang II‐infused wild‐type mice. In cultured human podocytes, Sirt6 knockdown exacerbated Ang II‐induced mitochondrial fission, whereas Sirt6 overexpression ameliorated the Ang II‐induced changes in the balance between mitochondrial fusion and fission. Functional studies revealed that Sirt6 deficiency exacerbated mitochondrial fission by promoting dynamin‐related protein 1 (Drp1) phosphorylation. Furthermore, Sirt6 mediated Drp1 phosphorylation by promoting Rho‐associated coiled coil‐containing protein kinase 1 (ROCK1) expression.ConclusionOur study has identified Sirt6 as a vital factor that protects against Ang II‐induced mitochondrial fission and apoptosis in podocytes via the ROCK1‐Drp1 signalling pathway.

FOXO4 alleviates hippocampal neuronal damage in epileptic mice via the miR-138-5p/ROCK2 axis.

Epilepsy (EP) is one of the most universal neurological disorders. This study investigated the mechanism of forkhead box protein O4 (FOXO4) on hippocampal neuronal damage in EP mice. Initially, the EP mouse model and the in vitro HT-22 cell model were established. EP seizures and neuronal damage in mice were assessed. FOXO4, microRNA (miR)-138-5p, and rho-associated coiled-coil containing protein kinase 2 (ROCK2) levels in hippocampal tissues or HT-22 cells were examined. The cell viability and apoptosis of HT-22 cells were determined. The concentrations of oxidative stress markers and the levels of inflammatory cytokines in hippocampal tissues or HT-22 cells were detected. We found that FOXO4 was poorly expressed in EP. FOXO4 overexpression alleviated hippocampal neuronal damage in EP mice and improved HT-22 cell viability and inhibited apoptosis, and decreased oxidative stress and inflammation in hippocampal tissue and HT-22 cells. The bindings of miR-138-5p to FOXO4 and ROCK2 were analyzed, which showed that FOXO4 promoted miR-138-5p via binding to the miR-138-5p promoter region, and miR-138-5p inhibited ROCK2 expression. Joint experiments showed that miR-138-5p suppression or ROCK2 overexpression reversed the alleviation of FOXO4 overexpression on hippocampal neuronal damage. FOXO4 inhibited ROCK2 expression via promoting miR-138-5p expression, thus alleviating hippocampal neuronal damage in EP mice.