Myosin Light Chain Phosphorylation Research Articles

Loss of prolyl hydroxylase 1 and 2 in SM22α-expressing cells prevents Hypoxia-Induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease characterized by increased vasoconstriction and vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) highly express the transcription factor hypoxia-inducible factor-1α (HIF-1α), yet the role of PASMC HIF-1α in the development of PAH remains controversial. To study the role of SMC HIF-1α in the pulmonary vascular response to acute and chronic hypoxia, we used a gain-of-function strategy to stabilize HIF-1α in PASMC by generating mice lacking prolyl hydroxylase domain (PHD) 1 and 2 in SM22α-expressing cells. This strategy increased HIF-1α expression and transcriptional activity under conditions of normoxia and hypoxia. Acute hypoxia increased right ventricular systolic pressure (RVSP) in control, but not in SM22α-PHD1/2-/- mice. Chronic hypoxia increased RVSP and vascular remodeling more in control SM22α-PHD1/2+/+ than in SM22α-PHD1/2-/- mice. In vitro studies demonstrated increased contractility and myosin light chain phosphorylation in isolated PHD1/2+/+ compared with PHD1/2-/- PASMC under both normoxic and hypoxic conditions. After chronic hypoxia, there was more p27 and less vascular remodeling in SM22α-PHD1/2-/- compared with SM22α-PHD1/2+/+ mice. Hypoxia increased p27 in PASMC isolated from control patients, but not in cells from patients with idiopathic pulmonary arterial hypertension (IPAH). These findings highlight an SM22α-expressing cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. Modulating HIF-1α expression in PASMC may represent a promising preventative and therapeutic strategy for patients with PAH.NEW & NOTEWORTHY In a mouse model wherein hypoxia-inducible factor 1 alpha (HIF-1α) is stabilized in vascular smooth muscle cells, we found that HIF-1α regulates vasoconstriction by limiting phosphorylation of myosin light chain and regulates vascular remodeling through p27 induction. These findings highlight a cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling.

Interaction between mitochondrial homeostasis and barrier function in lipopolysaccharide-induced endothelial cell injury.

This study aimed to investigate the effects of mitochondrial homeostasis on lipopolysaccharide (LPS)-induced endothelial cell barrier function and the mechanisms that underlie these effects. Cells were treated with LPS or oligomycin (mitochondrial adenosine triphosphate synthase inhibitor) and the mitochondrial morphology, mitochondrial reactive oxygen species (mtROS), and mitochondrial membrane potential (ΔΨm) were evaluated. Moreover, the shedding of glycocalyx-heparan sulphate (HS), the levels of HS-specific degrading enzyme heparanase (HPA), and the expression of occludin and zonula occludens (ZO-1) of Tight Junctions (TJ)s, which are mediated by myosin light chain phosphorylation (p-MLC), were assessed. Examining the changes in mitochondrial homeostasis showed that adding heparinase III, which is an exogenous HPA, can destroy the integrity of glycocalyx. LPS simultaneously increased mitochondrial swelling, mtROS, and ΔΨm. Without oligomycin effects, HS, HPA levels, and p-MLC were found to be elevated, and the destruction of occludin and ZO-1 increased. Heparinase III not only damaged the glycocalyx by increasing HS shedding but also increased mitochondrial swelling and mtROS and decreased ΔΨm. Mitochondrial homeostasis is involved in LPS-induced endothelial cell barrier dysfunction by aggravating HPA and p-MLC levels. In turn, the integrated glycocalyx protects mitochondrial homeostasis.

Potentiation of active force by cyclic strain in sheep carotid arterial smooth muscle.

The ability to generate force in large arteries is known to be augmented by cyclic strain that mimics the mechanically dynamic in vivo environment associated with blood pressure fluctuation experienced by these arteries. Cyclic strain does not induce a contractile response, like that observed in the myogenic response seen in small arteries, but prompts a substantial increase in the response to electrical stimulation. We coined this phenomenon "force potentiation." Because protein kinase C (PKC) and rho-kinase (ROCK) are known to play a role in increasing contractility of arterial smooth muscle by inhibition of myosin light chain phosphatase, and integrin-link kinase (ILK) is crucial in mechanotransduction, we examined how inhibition of these kinases affected force potentiation in sheep carotid artery. We found that phosphorylation of the regulatory myosin light chain was enhanced by cyclic strain, but the enhancement was observed only in activated, not in relaxed muscle. Inhibition of ROCK diminished force potentiation and active isometric force, likely due to the disinhibition of myosin light chain phosphatase. Inhibition of PKC abolished force potentiation without an effect on active force, suggesting a more exclusive role of PKC (compared with ROCK) in mediating force potentiation. Inhibition of ILK had a similar effect as PKC inhibition, suggesting that ILK may be an upstream kinase for PKC activation by mechanical stimuli. Taken together, the findings suggest that ILK, PKC, and ROCK are important kinases in the signal transduction pathway that mediate the effect of mechanical strain on force potentiation.NEW & NOTEWORTHY When subjected to mechanical strain, smooth muscle from large arteries has the ability to increase its force generation (force potentiation), which could be important in autoregulation of blood pressure. This phenomenon, however, does not involve a myogenic response, such as the one seen in small arteries and arterioles. Our work shows the involvement of ILK, PKC, and ROCK in the signal transduction pathway that mediates the force-potentiating effect of mechanical strain in large arteries.

Transcriptome analysis of the effects of maternal fructose exposure during gestation and lactation on the cytoskeleton of offspring hippocampus

Excessive fructose consumption poses a public health concern. Maternal fructose overconsumption impairs offspring hippocampal function, indicating hippocampal sensitivity to fructose. The hippocampus is the vital for spatial learning and memory, and the cytoskeletal structure is crucial for hippocampal neuronal plasticity. The Rho GTPase is a key regulator of neuronal cytoskeleton formation, with RhoA protein governing neuronal growth and development. The RhoA/ROCK2 pathway affects myosin light chain phosphorylation, leading to cytoskeletal reorganization, actin cytoskeleton changes, and neurite retraction. However, the effects of maternal fructose exposure during gestation and lactation on the offspring's neural cytoskeleton remain unclear. We established maternal fructose exposure models during gestation and lactation, assessing fasting blood glucose levels in offspring confirm model effectiveness. Whole transcriptome sequencing examined offspring hippocampal developmental programming. Immunofluorescence and Western blot analyses assessed RhoA/ROCK2 pathway expression changes in the hippocampus, with PCR validating transcriptome sequencing results. Transcriptome sequencing identified 193 and 298 differentially expressed genes in the control, 13% fructose, and 40% fructose groups, respectively. GO and KEGG analyses revealed significant changes related to neural development, learning memory, and cytoskeleton in both fructose-exposed groups. GSEA indicated substantial cytoskeleton-related alterations in the offspring. Immunofluorescence demonstrated significant CRMP2 protein down-regulation in both fructose-exposed groups. Western blotting showed significantly higher expression of RhoA, ROCK2, p-ROCK2, and p-MLC2 in the fructose-exposed groups (P < 0.05). Maternal high-fructose exposure significantly impacts gene expression in offspring. These alterations may influence hippocampal neurodevelopment by up-regulating the RhoA/ROCK2 pathway, leading to cytoskeletal damage and axon growth inhibition.

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs invitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI invivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

FOXO1 promotes endothelial cell elongation and angiogenesis by up-regulating the phosphorylation of myosin light chain 2.

The forkhead box O1 (FOXO1) is an important transcription factor related to proliferation, metabolism, and homeostasis, while the major phenotype of FOXO1-null mice is abnormal vascular morphology, such as vessel enlargement and dilation. In in vitro mouse embryonic stem cell (ESC)-differentiation system, Foxo1-/- vascular endothelial cells (ECs) fail to elongate, and mimic the abnormalities of FOXO1-deficiency in vivo. Here, we identified the PPP1R14C gene as the FOXO1 target genes responsible for elongating using transcriptome analyses in ESC-derived ECs (ESC-ECs), and found that the FOXO1-PPP1R14C-myosin light chain 2 (MLC2) axis is required for EC elongation during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP). PPP1R14C is an inhibitor of PP1, the catalytic subunit of MLCP. The abnormal morphology of Foxo1-/- ESC-ECs was associated with low level of PPP1R14C and loss of MLC2 phosphorylation, which were reversed by PPP1R14C-introduction. Knockdown of either FOXO1 or PPP1R14C suppressed vascular cord formation and reduced MLC2 phosphorylation in human ECs (HUVECs). The mouse and human PPP1R14C locus possesses an enhancer element containing conserved FOXO1-binding motifs. In vivo chemical inhibition of MLC2 phosphorylation caused dilated vascular structures in mouse embryos. Furthermore, foxo1 or ppp1r14c-knockdown zebrafish exhibited vascular malformations, which were also restored by PPP1R14C-introduction. Mechanistically, FOXO1 suppressed MLCP activity by up-regulating PPP1R14C expression, thereby promoting MLC2 phosphorylation and EC elongation, which are necessary for vascular development. Given the importance of MLC2 phosphorylation in cell morphogenesis, this study may provide novel insights into the role of FOXO1 in control of angiogenesis.

Delineating Zinc Influx Mechanisms during Platelet Activation.

Zinc (Zn2+) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn2+]o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn2+ influx are unknown. Fluctuations in intracellular zinc ([Zn2+]i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn2+]o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn2+ influx. [Zn2+]o stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 integrin. Platelet activation via GPVI or Zn2+ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn2+]i that were sensitive to the inhibition of Orai1, ZIP7, or IP3R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn2+]o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn2+ influx. Increases in [Zn2+]i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn2+ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn2+ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis.

Glycolytic activity is required for the onset of neural plate folding during neural tube closure in mouse embryos.

Physiological hypoxia is critical for placental mammalian development. However, the underlying mechanisms by which hypoxia regulates embryonic development remain unclear. We discovered that the expression of glycolytic genes partially depends on hypoxia in neuroepithelial cells of E8.25 mouse embryos. Consistent with this finding, inhibiting glycolysis during the early phase of neural tube closure (E8.0-8.5) resulted in a neural tube closure defect. In contrast, inhibiting the electron transport chain did not affect neural tube formation. Furthermore, inhibiting glycolysis affected cell proliferation, but not differentiation and survival. Inhibiting glycolysis repressed the phosphorylation of myosin light chain 2, and consequent neural plate folding. Our findings revealed that anaerobic glycolysis regulates neuroepithelial cell proliferation and apical constriction during the early phase of neural tube closure.

Internalization of apoptotic cells during efferocytosis requires Mertk-mediated calcium influx

Phagocytosis of apoptotic cells, called efferocytosis, requires calcium inside and outside of phagocytes. Due to its necessity, calcium flux is sophisticatedly modulated, and the level of intracellular calcium in phagocytes is ultimately elevated during efferocytosis. However, the role of elevated intracellular calcium in efferocytosis remains elusive. Here, we report that Mertk-mediated intracellular calcium elevation is necessary for internalization of apoptotic cells during efferocytosis. Drastic depletion of intracellular calcium abrogated the internalization step of efferocytosis by delaying phagocytic cup extension and closure. Especially, the defect of phagocytic cup closure for internalization of apoptotic cells was caused by impaired F-actin disassembly and the attenuated interaction of Calmodulin with myosin light chain kinase (MLCK), leading to diminished myosin light chain (MLC) phosphorylation. Genetic and pharmacological impairment of the Calmodulin-MLCK-MLC axis or Mertk-mediated calcium influx also resulted in inefficient efferocytosis due to a defect in internalization of the targets. Taken together, our observations imply that intracellular calcium elevation through Mertk-mediated calcium influx facilitates efferocytosis by inducing myosin II-mediated contraction and F-actin disassembly required for internalization of apoptotic cells.

Downregulation of connexin 43-based gap junctions underlies propofol-induced excessive relaxation in hypertensive vascular smooth muscle cells

BackgroundPostinduction hypotension caused by propofol remains a non-negligible problem for anesthesiologists, and is especially severe in chronic hypertensive patients with long-term vasoconstriction and decreased vascular elasticity. The functional change in gap junctions composed of Cx43 (Cx43-GJs) is reported as the biological basis of synchronized contraction or relaxation of blood vessels. Thus, we investigated the role of Cx43-GJs in propofol-induced dramatic blood pressure fluctuations in chronic hypertensive patients, and their internal mechanisms.MethodsHuman umbilical artery smooth muscle cells (HUASMCs) were pretreated with long-term angiotensin II (Ang II), with or without propofol, to simulate the contraction and relaxation of normal and hypertensive VSMCs during anesthesia induction. The levels of F-actin polymerization and MLC2 phosphorylation were used as indicators to observe the contraction and relaxation of HUASMCs. Different specific activators, inhibitors and siRNAs were used to explore the role of Cx43-GJs and Ca2+ as well as the RhoA/ LIMK2/cofilin and RhoA/MLCK signaling pathways in the contraction and relaxation of normal and hypertensive HUASMCs.ResultsBoth F-actin polymerization and MLC2 phosphorylation were significantly enhanced in Ang II-pretreated HUASMCs, along with higher expression of Cx43 protein and stronger function of Cx43-GJs than in normal HUASMCs. However, with propofol administration, similar to Gap26 and Cx43-siRNA, the function of Cx43-GJs in Ang II-pretreated HUASMCs was inhibited compared with that in normal HUASMCs, accompanied by a larger decrease in intracellular Ca2+ and the RhoA/LIMK2/cofilin and RhoA/MLCK signaling pathways. Eventually F-actin polymerization and MLC2 phosphorylation were more dramatically decreased. However, these effects could be reversed by RA with enhanced Cx43-GJ function.ConclusionLong-term exposure to Ang II significantly enhanced the expression of the Cx43 protein and function of Cx43-GJs in HUASMCs, resulting in the accumulation of intracellular Ca2+ and the activation of its downstream RhoA/LIMK2/cofilin and RhoA/MLCK signaling pathways, which maintained HUASMCs in a state of excessive-contraction. With inhibition of Cx43-GJs by propofol in Ang II-pretreated HUASMCs, intracellular Ca2+ and its downstream signaling pathways were dramatically inhibited, which ultimately excessively relaxed HUASMCs. This is the reason why the blood pressure fluctuation of patients with chronic hypertension was more severe after receiving propofol induction.9gEgDUsF4_-ddhb1iAhQGKVideo

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.

The intermediate filament protein nestin serves as a molecular hub for smooth muscle cytoskeletal signaling

BackgroundThe recruitment of the actin-regulatory proteins cortactin and profilin-1 (Pfn-1) to the membrane is important for the regulation of actin cytoskeletal reorganization and smooth muscle contraction. Polo-like kinase 1 (Plk1) and the type III intermediate filament protein vimentin are involved in smooth muscle contraction. Regulation of complex cytoskeletal signaling is not entirely elucidated. The aim of this study was to evaluate the role of nestin (a type VI intermediate filament protein) in cytoskeletal signaling in airway smooth muscle.MethodsNestin expression in human airway smooth muscle (HASM) was knocked down by specific shRNA or siRNA. The effects of nestin knockdown (KD) on the recruitment of cortactin and Pfn-1, actin polymerization, myosin light chain (MLC) phosphorylation, and contraction were evaluated by cellular and physiological approaches. Moreover, we assessed the effects of non-phosphorylatable nestin mutant on these biological processes.ResultsNestin KD reduced the recruitment of cortactin and Pfn-1, actin polymerization, and HASM contraction without affecting MLC phosphorylation. Moreover, contractile stimulation enhanced nestin phosphorylation at Thr-315 and the interaction of nestin with Plk1. Nestin KD also diminished phosphorylation of Plk1 and vimentin. The expression of T315A nestin mutant (alanine substitution at Thr-315) reduced the recruitment of cortactin and Pfn-1, actin polymerization, and HASM contraction without affecting MLC phosphorylation. Furthermore, Plk1 KD diminished nestin phosphorylation at this residue.ConclusionsNestin is an essential macromolecule that regulates actin cytoskeletal signaling via Plk1 in smooth muscle. Plk1 and nestin form an activation loop during contractile stimulation.

Mechanistic study for drug induced cholestasis using batch-fabricated 3D spheroids developed by agarose-stamping method

Cell spheroid culture can recapitulate the tissue microstructure and cellular responses in vivo. While there is a strong need to understand the modes of toxic action using the spheroid culture method, existing preparation techniques suffer from low efficiency and high cost. Herein, we developed a metal stamp containing hundreds of protrusions for batch bulk preparation of cell spheroids in each well of the culture plates. The agarose matrix imprinted by the stamp can form an array of hemispherical pits, which facilitated the fabrication of hundreds of uniformly sized rat hepatocyte spheroids in each well. Chlorpromazine (CPZ) was used as a model drug to investigate the mechanism for drug induced cholestasis (DIC) by agarose-stamping method. Hepatocyte spheroids showed a more sensitive detection of hepatotoxicity compared to 2D and Matrigel-based culture systems. Cell spheroids were also collected for staining of cholestatic protein and showed a CPZ-concentration-dependent decrease of bile acid efflux related proteins (BSEP and MRP2) and tight junction (ZO-1). In addition, the stamping system successfully delineated the DIC mechanism by CPZ that may be associated with the phosphorylation of MYPT1 and MLC2, two central proteins in the Rho-associated protein kinase pathway (ROCK), which were significantly attenuated by ROCK inhibitors. Our results demonstrated a large-scale fabrication of cell spheroids by the agarose-stamping method, with promising benefits for exploring the mechanisms for drug hepatotoxic responses.

Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart

Myosin functions as the “molecular motor” of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an ‘atrial’ and ‘ventricular’ isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

Histone Modification of Osteogenesis Related Genes Triggered by Substrate Topography Promotes Human Mesenchymal Stem Cell Differentiation.

The clinical success of orthopedic implants is closely related to their integration in the bone tissue promoted by rough device surfaces. The biological response of precursor cells to their artificial microenvironments plays a critical role in this process. In this study, we elucidated the relation between cell instructivity and surface microstructure of polycarbonate (PC)-based model substrates. The rough surface structure (hPC) with an average peak spacing (Sm) similar to the trabecular spacing of trabecular bone improved osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), as compared to the smooth surface (sPC) and the surface with a moderate Sm value (mPC). The hPC substrate promoted the cell adhesion and assembling of F-actin and enhanced cell contractile force by upregulating phosphorylated myosin light chain (pMLC) expression. The increased cell contractile force led to YAP nuclear translocation and the elongation of cell nuclei, presenting higher levels of active form of Lamin A/C. The nuclear deformation alternated the histone modification profile, particularly the decrease of H3K27me3 and increase of H3K9ac on the promoter region of osteogenesis related genes (ALPL, RUNX2, and OCN). Mechanism study using inhibitors and siRNAs elucidated the role of YAP, integrin, F-actin, myosin, and nuclear membrane proteins in such a regulatory process of surface topography on stem cell fate. These mechanistical insights on the epigenetic level give a new perspective in understanding of the interaction of substrate and stem cells as well as provide valuable criteria for designing bioinstructive orthopedic implants.

THE INHIBITORY EFFECTS OF VALPROIC ACID ON RHOA-MEDIATED VASCULAR SMOOTH MUSCLE CELL CONTRACTION

Objective: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. Growing evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of VPA on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which VPA inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Design and method: We performed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), RhoA activity assay, transfection of constitutively active (CA)-RhoA gene, and western blot analyses. We also conducted ex vivo studies using isolated rat aortas. Results: VPA inhibited phenylephrine (PE)-induced phosphorylation of myosin light chain (MLC) at Ser19 (p-MLC-Ser19) in VSMCs. VPA did not alter expressions of MLC kinase and Rho-associated kinase 1, the upstream kinases responsible for MLC phosphorylation. In addition, VPA did not affect phosphorylation of myosin phosphatase target subunit 1, a regulatory subunit of myosin phosphatase. VPA decreased RhoA mRNA expression and its protein levels in a time- and dose-dependent manner, and as expected, VPA decreased amount of active GTP-bound RhoA in a dose-dependent manner. Ectopic expression of CA-RhoA gene significantly reversed VPA-inhibited p-MLC-Ser19 and p-CPI-17-Thr38. Finally, VPA significantly attenuated PE-induced vessel contraction in endothelium-deprived rat aortas, and similar to the in vitro results, VPA decreased RhoA expression and levels of p-MLC-Ser19 and p-CPI-17-Thr38 in rat aortas. Conclusions: We demonstrate that VPA mitigates PE-induced VSMC contractility and vessel contraction by decreasing expressions of RhoA and p-MLC-Ser19 in rat VSMCs and aortas. These results suggest that VPA may be useful in the treatment of vasospastic diseases including coronary artery vasospasm and post-subarachnoid hemorrhage cerebral vasospasm.

SALT-INDUCED VASCULAR DYSFUNCTION INVOLVES REDOX ACTIVATION OF PARP/TRPM2 SIGNALLING AND INFLAMMASOME ASSEMBLY

Objective: Many individuals with essential hypertension are salt sensitive. High salt diet (HSD) has deleterious effects on the vasculature by mechanisms not fully elucidated. We explored molecular mechanisms, focusing on pro-inflammatory pathways whereby HSD influences vascular function in hypertension focusing on PARP-regulated TRPM2, a redox-sensitive Ca2+ channel, and the inflammasome. In particular we assessed if HSD induces a pro-oxidant environment leading to PARP-induced TRPM2-activation, Ca2+ influx and inflammasome assembly, leading to vascular damage and hypertension. Design and method: C57BL/6 mice were treated with normal or 4% high salt diet (HSD) for 5 weeks. Vascular function/structure was assessed in mesenteric arteries mounted in wire/pressure myography. VSMCs from rodents and humans were exposed to normal (145 mM) and high salt medium (180 mM) (HSM). Results: In presence of HSM, VSMCs present increased ROS generation (Control:0.7033±0.05 vs HSM:1.243±0.09) and PARP activation (Control: 94.25±6.67 vs HSM: 369.6±79.46), effect no longer observed in presence of ROS scavenger tiron, and antioxidant PEG-catalase. HSM induces Ca2+ influx in VSMCs (Control: 25562±880.48 vs HSM: 30925±1263.85), effect reversed by tiron, PARP inhibitor (olaparib) and TRPM2 blocker (8-br). NLRP3 activation (IL-1beta and Il-18 production) in VSMCs is increased by HSM (IL18 - Control: 93.82±1.97 vs HSM:127.30±6.57; IL-1 beta Control: 99.86±2.49 vs HSM: 125.30±3.43) HSM, effect followed by myosin light chain phosphorylation, which is dependent on ROS, PARP and TRPM2 activation. Animals treated with HSD present increased systolic BP (Control: 121.3±2.15 vs HSD: 134.90±3.04), outward hypertrophic remodelling and increased vascular contractility, effect reversed by PARP and NLRP3 inhibitors. Conclusions: In conclusion, HSD-induced hypertension involves ROS generation leading to activation of PARP-TRPM2 axis, increased calcium influx, NLRP3 activation and consequent vascular hypercontractility. We define novel pathways linking HSD to pathophysiological mechanisms underlying salt-sensitive hypertension.

Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function

Fetal lung fibroblasts contribute dynamic infrastructure for the developing lung. These cells undergo dynamic mechanical transitions, including cyclic stretch and spreading, which are integral to lung growth in utero. We investigated the role of the nuclear envelope protein emerin in cellular responses to these dynamic mechanical transitions. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, fibroblasts that were acutely downregulated for emerin showed incomplete reorientation of both nuclei and actin cytoskeleton. Emerin-downregulated fibroblasts were also aberrantly circular in contrast to the spindle-shaped controls and exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus. Emerin knockdown was also associated with reduced myosin light chain phosphorylation during cell spreading. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and production. These findings indicate that nuclear-cytoskeletal coupling serves a role in the dynamic regulation of cytoskeletal structure and function and may also impact the transmission of traction force to the extracellular matrix microenvironment.