Modulation by thyroid hormone of myosin light chain phosphorylation and aquaporin 5 protein expression in intact lung

Myosin light chain kinase (MLCK) may play a key role in cellular contraction, paracellular permeability and lung water homeostasis. In vitro, thyroid hormones (THs) potently inhibit MLCKactivation and, hence, MLC phosphorylation. Whether similar effect is exerted by THs in in vivo systems is not known. Therefore, we investigated the effects of hypothyroid (HO) and hyperthyroid (HR) states on the level of phospho-MLC, aquaporin 5 (AQP5) protein expression, and water holding capacity in the rat lung. Alterations in thyroid state were induced by adding methimazole or levothyroxine (L-T4) to animals’ drinking water. Serum TH concentration and thyroid gland histomorphology were assessed to verify the onset of the thyroid state. Lung phospho-MLC and AQP5 proteins were assessed by Western blotting and immunohistochemistry. Lung extravascular water content was estimated by the tissue wet weight-to-dry weight (W/D) ratio. The HO state induced significant increases in the expression of lung phospho-MLC and AQP5 proteins. In contrast, the HR state caused moderate decreases in lung phospho-MLC and AQP5 proteins. While lung water holding capacity was significantly increased in HO animals, it was significantly reduced in HR animals. The data of this study show that THs are able to modulate MLC phosphorylation in in vivo systems. Besides, they suggest that the circulating level of THs may alter lung fluid balance not only through expression of water channels but also through regulation of cellular contraction and paracellular permeability

A bioinformatic and computational study of myosin phosphatase subunit diversity.

Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression.

Increased activation of the Rho-A/Rho-kinase pathway in the renal vascular system is responsible for the enhanced reactivity to exogenous vasopressin in endotoxemic rats.

OBJECTIVE: We evaluated the role of the renal vascular system and the Rho-A/Rho-kinase pathway in the maintenance of the pressor effects of vasopressin in endotoxemic rats. DESIGN: In vitro and in vivo animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats (200-300 g). INTERVENTION: Rats received either saline or lipopolysaccharide (10 mg/kg, intraperitoneal) 6 or 24 hours before the experiments. The effects of vasopressin on isolated aortic rings, cardiac function, mean arterial pressure, and both the renal vascular perfusion pressure of perfused kidneys in vitro and renal blood flow in situ were evaluated. The role of Rho-kinase in the renal and systemic effects of vasopressin was investigated through administration of the selective inhibitor Y-27632 and Western blot analysis. MEASUREMENTS AND MAIN RESULTS: The effect of vasopressin on mean arterial pressure was unaltered and that on renal vascular perfusion pressure enhanced in endotoxemic rats at both 6 and 24 hours after lipopolysaccharide, despite reduced contractile responses in aortic rings and the lack of effect on cardiac function. Vasopressin (3, 10, and 30 pmol/kg, IV) produced increased reduction in renal blood flow in endotoxemic rats. In perfused kidneys from lipopolysaccharide groups, administration of Y-27632 reverted the hyperreactivity to vasopressin. Treatment with Y-27632 partially inhibited the effects of vasopressin on mean arterial pressure and significantly reduced the effects of vasopressin on renal blood flow in control but not in endotoxemic rats. Although the protein levels of Rho-A and Rho-kinase I and II had not been impaired, the levels of phosphorylated myosin phosphatase-targeting subunit 1, the regulatory subunit of myosin phosphatase that is inhibited by Rho-kinase, were increased in both the renal cortex and the renal medulla of endotoxemic rats. CONCLUSION: Our data suggest that activation of Rho-kinase potentiates the vascular effects of vasopressin in the kidneys, contributing to the maintenance of the hypertensive effects of this agent during septic shock.

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM).

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT(2A) receptor, 5-HT(1B) receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr(696))-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.

Deficiency in myosin light-chain phosphorylation causes cytokinesis failure and multipolarity in cancer cells.

Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which are an important part of malignant transformation in the most recent model of tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. In this study, we show that cancer cells often failed at cytokinesis because of decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light-chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light-chain phosphorylation was restored to normal levels by phosphatase knockdown, multinucleation and multipolar mitosis were markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light-chain kinase in nonmalignant cells could recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating that these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells.

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.