Kaempferol mitigates sepsis-induced acute lung injury by modulating the SphK1/S1P/S1PR1/MLC2 signaling pathway to restore the integrity of the pulmonary endothelial cell barrier.

Sepsis-induced acute lung injury (SALI) is the common complication of sepsis, resulting in high incidence and mortality rates. The primary pathogenesis of SALI is the interplay between acute inflammation and endothelial barrier damage. Studies have shown that kaempferol (KPF) has anti-sepsis properties. Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway's significance in acute lung damage and S1P receptor 1 (S1PR1) agonists potential in myosin light chain 2 (MLC2) phosphorylation are documented. Whether KPF can regulate the SphK1/S1P/S1PR1/MLC2 signaling pathway to protect the lung endothelial barrier remains unclear. This study investigates the KPF's therapeutic effects and molecular mechanisms in repairing endothelial cell barrier damage in both LPS-induced sepsis mice and human umbilical vein endothelial cells (HUVECs). KPF significantly reduced lung tissue damage and showed anti-inflammatory effects by decreasing IL-6 and TNF-α synthesis in the sepsis mice model. Further, KPF administration can reduce the high permeability of the LPS-induced endothelial cell barrier and alleviate lung endothelial cell barrier injury. Mechanistic studies showed that KPF pretreatment can suppress MLC2 hyperphosphorylation and decrease SphK1, S1P, and S1PR1 levels. The SphK1/S1P/S1PR1/MLC2 signaling pathway controls the downstream proteins linked to endothelial barrier damage, and the Western blot (WB) showed that KPF raised the protein levels. These proteins include zonula occludens (ZO)-1, vascular endothelial (VE)-cadherin and Occludin. The present work revealed that in mice exhibiting sepsis triggered by LPS, KPF strengthened the endothelial barrier and reduced the inflammatory response. The SphK1/S1P/S1PR1/MLC2 pathway's modulation is the mechanism underlying this impact.

Investigating skeletal muscle biomarkers for the early detection of Australian myotoxic snake envenoming: an animal model pilot study.

INTRODUCTION: Myotoxicity is an important toxidrome that can occur with envenoming from multiple Australian snake types. Early antivenom administration is an important strategy to reduce the incidence and severity of myotoxicity. The current gold standard biomarker, serum creatine kinase activity, does not rise early enough to facilitate early antivenom administration. Several other skeletal muscle biomarkers have shown promise in other animal models and scenarios. The aim of this study was to examine the predictive values of six skeletal muscle biomarkers in a rat model of Australian snake myotoxicity. METHODS: Sprague-Dawley rats were anaesthetised and administered either Pseudechis porphyriacus (red-bellied black snake) or Notechis scutatus (tiger snake) venom, or normal saline via intramuscular injection. Blood samples were collected. Assays were performed for serum creatine kinase skeletal muscle troponin-I concentration, skeletal muscle troponin-C concentration, myoglobin activity, skeletal muscle myosin light chain-1 concentration, and creatine kinase-MM activity. Serum markers were plotted against time, with comparison of area under the concentration (or activity)-time curve. The predictive values of six skeletal muscle biomarkers were examined using receiver operating characteristic curves. RESULTS: There was no difference in area under the serum creatine kinase activity-time curve between venom and control groups. Serum creatine kinase-MM activity rose early in the venom treated rats, which had a significantly greater area under the serum activity-time curve. No difference in area under the serum concentration-time curve was demonstrated for the other biomarkers. Creatine kinase-MM activity had a superior predictive values than creatine kinase activity at 0-4 hours and 0-10 hours after venom administration, as indicated by area under the receiver operating characteristic curves (95 per cent confidence intervals) of 0.91 (0.78-1.00) and 0.88 (0.73-1.00) versus 0.79 (0.63-0.95) and 0.66 (0.51-0.80). DISCUSSION: The limitations of serum creatine kinase activity in early detection of myotoxicity were demonstrated in this rat model. CONCLUSION: Serum creatine kinase-MM activity was superior for early detection of Australian myotoxic snake envenoming.

Pharmacological control of angiogenesis by regulating phosphorylation of myosin light chain 2.

BACKGROUND: Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis. METHODS AND RESULTS: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs. CONCLUSION: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.

Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle.

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the "pCa-velocity" relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

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Objective: To investigate the synergistic regulation of the polarization of mesenchymal stem cells by integrin and N-cadherin-mediated mechanical adhesion and the underlying mechanobiological mechanisms. Methods: Bilayer polyethylene glyeol (PEG) hydrogels were formulated and modified with RGD and HAVDI peptides, respectively, to achieve mechanical adhesion to integrin and N-cadherin and to replicate the integrin-mediated mechanical interaction between cells and the extracellular matrix and the N-cadherin-mediated cell-cell mechanical interaction. The polar proteins, phosphatidylinositol 3-kinase (PI3K) and phosphorylated myosin light chain (pMLC), were characterized through immunofluorescence staining in individual cells with or without contact with HAVDI peptides under integrin-mediated adhesion, N-cadherin-mediated adhesion, and different intracellular forces. Their expression levels and polar distribution were analyzed using Image J. Results: Integrin-mediated adhesion induced significantly higher polar strengths of PI3K and pMLC in the contact group than in those in the no contact group, resulting in the concentration of the polar angle of PI3K to ß-catenin in the range of 135° to 180° and the concentration of the polar angle of pMLC to ß-catenin in the range of 0° to 45° in the contact group. Inhibition of integrin function led to inhibition of the polarity distribution of PI3K in the contact group, but did not change the polarity distribution of pMLC protein. The effect of N-cadherin on the polarity distributions of PI3K and pMLC was similar to that of integrin. However, inhibition of the mechanical adhesion of N-cadherin led to inhibition of the polarity intensity and polarity angle distribution of PI3K and pMLC proteins in the contact group. Furthermore, inhibition of the mechanical adhesion of N-cadherin caused weakened polarity intensity of integrin ß1, reducing the proportion of cells with polarity angles between integrin ß1 and ß-catenin concentrating in the range of 135° to 180°. Additionally, intracellular forces influenced the polar distribution of PI3K and pMLC proteins. Reducing intracellular forces weakened the polarity intensity of PI3K and pMLC proteins and their polarity distribution, while increasing intracellular forces enhanced the polarity intensity of PI3K and pMLC proteins and their polarity distribution. Conclusion: Integrin and N-cadherin co-regulate the polarity distribution of cell proteins and N-cadherin can play an important role in the polarity regulation of stem cells through local inhibition of integrin.

Lifibrate attenuates blood-brain barrier damage following ischemic stroke via the MLCK/p-MLC/ZO-1 axis.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro. In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier.

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

Ablation of skeletal muscle estrogen receptor alpha impairs contractility in male mice.

Estradiol and estrogen receptor α (ERα) have been shown to be important for the maintenance of skeletal muscle strength in females; however, little is known about the roles of estradiol and ERα in male muscle. The purpose of this study was to determine if skeletal muscle ERα is required for optimal contractility in male mice. We hypothesize that reduced ERα in skeletal muscle impairs contractility in male mice. Skeletal muscle-specific knockout (skmERαKO) male mice exhibited reduced strength across multiple muscles and several contractile parameters related to force generation and kinetics compared with wild-type littermates (skmERαWT). Isolated EDL muscle-specific isometric tetanic force, peak twitch force, peak concentric and peak eccentric forces, as well as the maximal rates of force development and relaxation were 11%-21% lower in skmERαKO compared with skmERαWT mice. In contrast, isolated soleus muscles from skmERαKO mice were not affected. In vivo peak torque of the anterior crural muscles was 20% lower in skmERαKO compared with skmERαWT mice. Muscle masses, contractile protein contents, fiber types, phosphorylation of the myosin regulatory light chain, and caffeine-elicited force did not differ between muscles of skmERαKO and skmERαWT mice, suggesting that strength deficits were not due to size, composition, or calcium release components of muscle contraction. These results indicate that in male mice, reduced skeletal muscle ERα blunts contractility to a magnitude similar to that previously reported in females; however, the mechanism may be sexually dimorphic.NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo contractility of leg muscles with reduced estrogen receptor α (ERα) in male mice and reported that force generation and contraction kinetics are impaired. In contrast to findings in females, phosphorylation of myosin regulatory light chain cannot account for low force production in male skeletal muscle ERα knockout mice. These results indicate that ERα is required for optimal contractility in males and females but via sexually dimorphic means.

The MYPT2-regulated striated muscle-specific myosin light chain phosphatase limits cardiac myosin phosphorylation in vivo.

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cardiac myosin light chain kinase and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC-phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of ß-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy.

Myosin light chain 6 (Myl6) interacts with kindlin-3 and is required to support integrin αIIbß3 activation in platelets in mice.

BACKGROUND: Kindlin-3 in platelets plays an essential role in supporting integrin αIIbß3 activation, platelet spreading, aggregation, and clot retraction by binding to the integrin ß3 cytoplasmic tail. However, the mechanism by which kindlin-3 mediates the crosstalk between integrin αIIbß3 and myosin in platelets remains unknown. OBJECTIVES: To examine the role of myosin light chain 6 (Myl6) in supporting integrin αIIbß3 activation in platelets. METHODS: Myl6fl/flPF4-Cre mice with a deficiency of Myl6 in the megakaryocyte lineage were generated, and integrin αIIbß3 activation in Myl6-deficient platelets was analyzed. RESULTS: We identified a novel kindlin-3 binding protein, Myl6, an essential light chain of myosin in platelets. Myl6fl/flPF4-Cre mice exhibited significant macrothrombocytopenia resulting from defective proplatelet formation. In the absence of Myl6, integrin αIIbß3 activation in platelets was significantly suppressed, and platelet aggregation was substantially impaired. Interestingly, the deficiency of Myl6 in platelets preferentially affected the binding of a multivalent ligand compared to a monovalent ligand to integrin αIIbß3 upon activation, indicating that Myl6 may contribute to the avidity modulation of integrin αIIbß3 by binding to kindlin-3. Furthermore, blood coagulation ability was impaired in Myl6fl/flPF4-Cre mice, and consistently, these mice exhibited defects in both hemostatic and thrombotic functions. CONCLUSION: In summary, these results suggest that Myl6, as a novel kindlin-3 binding partner, is required to support integrin αIIbß3 activation in platelets, which plays an important role in both hemostasis and thrombosis.

Functional characterization of calmodulin-like proteins, CML13 and CML14, as novel light chains of Arabidopsis class VIII myosins.

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-ß induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-ß significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-ß stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-ß significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-ß. ZIPK gene knockdown (KD) also significantly reduced TGF-ß-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-ß-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

MRLC controls apoptotic cell death and functions to regulate epidermal development during planarian regeneration and homeostasis.

Adult stem cells (ASCs) are pluripotent cells with the capacity to self-renew and constantly replace lost cells due to physiological turnover or injury. Understanding the molecular mechanisms of the precise coordination of stem cell proliferation and proper cell fate decision is important to regeneration and organismal homeostasis. The planarian epidermis provides a highly tractable model to study ASC complex dynamic due to the distinct spatiotemporal differentiation stages during lineage development. Here, we identified the myosin regulatory light chain (MRLC) homologue in the Dugesia japonica transcriptome. We found high expression levels of MRLC in wound region during regeneration and also expressed in late epidermal progenitors as an essential regulator of the lineage from neoblasts to mature epidermal cells. We investigated the function of MRLC using in situ hybridization, real-time polymerase chain reaction and double fluorescent and uncovered the potential mechanism. Knockdown of MRLC leads to a remarkable increase in cell death, causes severe abnormalities during regeneration and homeostasis and eventually leads to animal death. The global decrease in epidermal cell in MRLC RNAi animals induces accelerated epidermal proliferation and differentiation. Additionally, we find that MRLC is co-expressed with cdc42 and acts cooperatively to control the epidermal lineage development by affecting cell death. Our results uncover an important role of MRLC, as an inhibitor of apoptosis, involves in epidermal development.

LAMB3 Promotes Myofibrogenesis and Cytoskeletal Reorganization in Endometrial Stromal Cells via the RhoA/ROCK1/MYL9 Pathway.

LAMB3, a major extracellular matrix and basal membrane component, is involved in wound healing. We aimed to understand its role in Asherman's syndrome (AS), which is associated with infertility, by using bioinformatics analysis and cultured endometrial stromal cells (ESCs). MRNAs extracted from tissues obtained from control subjects and patients with severe intrauterine adhesion were sequenced and subjected to bioinformatics analysis and the RhoA/ROCK1/MYL9 pathway was implicated and this subsequently studied using cultured primary ESCs. The effects of overexpression and knockdown and activation and inhibition of LAMB3 on the mesenchymal to myofibroblastic phenotypic transformation of ECCs were assessed using PCR and western blot analysis. Phalloidin was used to localize the actin cytoskeletal proteins. Silencing of LAMB3 reversed the TGF-ß-induced ESC myofibroblast phenotype conversion, whereas overexpression of LAMB3 promoted this process. Activation and silencing of LAMB3 led to remodeling of the ESC cytoskeleton. Overexpression and silencing of LAMB3 caused activation and inhibition of ESCs, respectively. Y-27632 and LPA reversed the activation and inhibition of the RhoA/ROCK1/MYL9 pathway after overexpression and silencing, respectively. These results suggest that LAMB3 can regulate ESC fibrosis transformation and cytoskeleton remodeling via the RhoA/ROCK1/MYL9 pathway. This study provides a potential new target for gene therapy and drug intervention of AS.

Role of protein kinase D1 in vasoconstriction and haemodynamics in rats.

AIMS: Protein kinase D (PKD), once considered an effector of protein kinase C (PKC), now plays many pathophysiological roles in various tissues. However, little is known about role of PKD in vascular function. We investigated the role of PKD in contraction of rat aorta and human aortic smooth muscle cells (HASMCs) and in haemodynamics in rats. METHODS AND RESULTS: Isometric tension of rat aortic was measured to examine norepinephrine-induced contraction in the presence of PKD, PKC and Rho-kinase inhibitors. Phosphorylation of PKD1, myosin targeting subunit-1 (MYPT1), myosin light chain (MLC), CPI-17 and heat-shock protein 27 (HSP27), and actin polymerization were measured in the aorta. Phosphorylation of MYPT1 and MLC was also measured in HASMCs knocked down with specific siRNAs of PKD 1, 2 and 3. Intracellular calcium concentrations and cell shortening were measured in HASMCs. Norepinephrine-induced aortic contraction was accompanied by increased phosphorylation of PKD1, MYPT1 and MLC and actin polymerization, all of which were attenuated with PKD inhibitor CRT0066101. PKD1 phosphorylation was not inhibited by PKC inhibitor, chelerythrine or Rho kinase inhibitor, fasudil. In HASMCs, the phosphorylation of MYPT1 and MLC was attenuated by PKD1, but not PKD2, 3 knockdown. In HASMCs, CRT0066101 inhibited norepinephrine-induced cell shortening without affecting calcium concentration. Administration of CRT0066101 decreased systemic vascular resistance and blood pressure without affecting cardiac output in rats. CONCLUSIONS: PKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner.

Inhibition of UTX/KDM6A improves recovery of spinal cord injury by attenuating BSCB permeability and macrophage infiltration through the MLCK/p-MLC pathway.

Spinal cord injury (SCI) can prompt an immediate disruption to the blood-spinal cord barrier (BSCB). Restoring the integrity of this barrier is vital for the recovery of neurological function post-SCI. The UTX protein, a histone demethylase, has been shown in previous research to promote vascular regeneration and neurological recovery in mice with SCI. However, it is unclear whether UTX knockout could facilitate the recovery of the BSCB by reducing its permeability. In this study, we systematically studied BSCB disruption and permeability at different time points after SCI and found that conditional UTX deletion in endothelial cells (ECs) can reduce BSCB permeability, decrease inflammatory cell infiltration and ROS production, and improve neurological function recovery after SCI. Subsequently, we used RNA sequencing and ChIP-qPCR to confirm that conditional UTX knockout in ECs can down-regulate expression of myosin light chain kinase (MLCK), which specifically mediates myosin light chain (MLC) phosphorylation and is involved in actin contraction, cell retraction, and tight junctions (TJs) protein integrity. Moreover, we found that MLCK overexpression can increase the ratio of p-MLC/MLC, further break TJs, and exacerbate BSCB deterioration. Overall, our findings indicate that UTX knockout could inhibit the MLCK/p-MLC pathway, resulting in decreased BSCB permeability, and ultimately promoting neurological recovery in mice. These results suggest that UTX is a promising new target for treating SCI.

MYL9 promotes squamous cervical cancer migration and invasion by enhancing aerobic glycolysis.

OBJECTIVE: This study explored the mechanism of squamous cervical cancer (SCC) progression. METHODS: Reverse transcription-quantitative polymerase chain reaction and western blotting were used to evaluate the expression of myosin light chain 9 (MYL9) in SCC tissues and cell lines. Furthermore, Transwell and Boyden assays were used to assess the function of MYL9 in SCC progression. In addition, the levels of lactate and aerobic glycolysis were used to explore the detailed mechanism of MYL9 in SCC. RESULTS: The mRNA and protein levels of MYL9 were elevated in SCC tissues, and MYL9 knockdown inhibited the migration and invasion of SCC cell lines. A mechanistic study demonstrated that MYL9 promotes SCC migration and invasion by enhancing aerobic glycolysis and increasing the activity of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. CONCLUSIONS: MYL9 was upregulated in SCC, and it enhanced JAK2/STAT3 pathway activity and promoted metastasis and glycolysis in SCC.