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PloS One 2015Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth...
Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.
Topics: Animals; Carbachol; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gastric Fundus; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Contraction; Muscle Relaxation; Muscle Tonus; Muscle, Smooth; Myosin Light Chains; Myosin-Light-Chain Kinase; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Peptide Fragments; Phosphorylation; Potassium Chloride
PubMed: 26258553
DOI: 10.1371/journal.pone.0134876 -
Nature Reviews. Cancer Jul 2014Metastasis is the leading cause of cancer-related deaths, but it is unclear how cancer cells escape their primary sites in epithelia and disseminate to other sites in... (Review)
Review
Metastasis is the leading cause of cancer-related deaths, but it is unclear how cancer cells escape their primary sites in epithelia and disseminate to other sites in the body. One emerging possibility is that transformed epithelial cells could invade the underlying tissue by a process called cell extrusion, which epithelia use to remove cells without disrupting their barrier function. Typically, during normal cell turnover, live cells extrude apically from the epithelium into the lumen and later die by anoikis; however, several oncogenic mutations shift cell extrusion basally, towards the tissue that the epithelium encases. Tumour cells with high levels of survival and motility signals could use basal extrusion to escape from the tissue and migrate to other sites within the body.
Topics: Cell Movement; Epithelial Cells; Humans; Neoplasm Invasiveness; Neoplasms
PubMed: 24943812
DOI: 10.1038/nrc3767 -
The EMBO Journal Aug 2020Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts...
Plasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. We show that apical endocytosis and late endosome-mediated trafficking are required for membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically and then continuously endocytosed, together with apical and basal cargo. We describe an organelle carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis and which we suggest acts as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments.
Topics: Animals; Cell Membrane; Drosophila melanogaster; Endosomes; Organogenesis; Transcytosis
PubMed: 32657472
DOI: 10.15252/embj.2020105332 -
Biology Open Nov 2018Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction...
Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3β overcomes knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of , suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
PubMed: 30305282
DOI: 10.1242/bio.034520 -
Current Biology : CB May 2022As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the...
As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination, mechanical feedback, perhaps via adherens junctions, has been suggested to play a role. However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture. In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM), which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis. To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.
Topics: Cell Cycle; Cell Nucleus; Epithelium; Mechanotransduction, Cellular; Mitosis
PubMed: 35338851
DOI: 10.1016/j.cub.2022.03.004 -
Biomolecules Oct 2020When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a... (Comparative Study)
Comparative Study
When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.
Topics: Animals; Animals, Newborn; Cell Differentiation; Cells, Cultured; Connexin 43; Connexins; Gap Junctions; Gene Knockdown Techniques; Keratinocytes; Mice; Mice, Inbred C57BL; Rats; Rodentia; Skin
PubMed: 33066499
DOI: 10.3390/biom10101443 -
Frontiers in Cell and Developmental... 2020During the development of the cortex, newly generated neurons migrate long-distances in the expanding tissue to reach their final positions. Pyramidal neurons are... (Review)
Review
During the development of the cortex, newly generated neurons migrate long-distances in the expanding tissue to reach their final positions. Pyramidal neurons are produced from dorsal progenitors, e.g., radial glia (RGs) in the ventricular zone, and then migrate along RG processes basally toward the cortex. These neurons are hence dependent upon RG extensions to support their migration from apical to basal regions. Several studies have investigated how intracellular determinants are required for RG polarity and subsequent formation and maintenance of their processes. Fewer studies have identified the influence of the extracellular environment on this architecture. This review will focus on extracellular factors which influence RG morphology and pyramidal neuronal migration during normal development and their perturbations in pathology. During cortical development, RGs are present in different strategic positions: apical RGs (aRGs) have their cell bodies located in the ventricular zone with an apical process contacting the ventricle, while they also have a basal process extending radially to reach the pial surface of the cortex. This particular conformation allows aRGs to be exposed to long range and short range signaling cues, whereas basal RGs (bRGs, also known as outer RGs, oRGs) have their cell bodies located throughout the cortical wall, limiting their access to ventricular factors. Long range signals impacting aRGs include secreted molecules present in the embryonic cerebrospinal fluid (e.g., Neuregulin, EGF, FGF, Wnt, BMP). Secreted molecules also contribute to the extracellular matrix (fibronectin, laminin, reelin). Classical short range factors include cell to cell signaling, adhesion molecules and mechano-transduction mechanisms (e.g., TAG1, Notch, cadherins, mechanical tension). Changes in one or several of these components influencing the RG extracellular environment can disrupt the development or maintenance of RG architecture on which neuronal migration relies, leading to a range of cortical malformations. First, we will detail the known long range signaling cues impacting RG. Then, we will review how short range cell contacts are also important to instruct the RG framework. Understanding how RG processes are structured by their environment to maintain and support radial migration is a critical part of the investigation of neurodevelopmental disorders.
PubMed: 33178693
DOI: 10.3389/fcell.2020.578341 -
Journal of the Endocrine Society Aug 2022Preparation of patients with iodine contrast media (ICM) allergy who require adrenal vein sampling (AVS) to establish source of aldosterone excess of their confirmed...
CONTEXT
Preparation of patients with iodine contrast media (ICM) allergy who require adrenal vein sampling (AVS) to establish source of aldosterone excess of their confirmed primary aldosteronism (PA) is controversial. Usual premedication with high-dose prednisone can interfere with cortisol determinations, possibly altering the aldosterone to cortisol ratios for the identification of lateralized aldosterone excess.
OBJECTIVE
We aimed to evaluate the efficacy and safety of premedication with high-dose dexamethasone to perform AVS in patients with ICM.
METHODS
One hundred and seventy-seven consecutive patients with confirmed PA who underwent bilateral simultaneous basal and post-ACTH bolus AVS at our center between January 2010 and December 2020 were retrospectively analyzed for history of ICM allergy. A total of 7 patients (4%) with previous allergic reactions to ICM were prepared with 3 doses of 7.5 mg dexamethasone premedication rather than the usual 50 mg of prednisone.
RESULTS
No breakthrough allergic reactions were reported in the 7 patients. Despite adequate serum cortisol suppression following dexamethasone, the basal and post-ACTH selectivity index were respectively > 2 and > 5 bilaterally in all patients, confirming adequate cannulation of both adrenal veins. Four patients had lateralized ratios (A/C ratio > 2 basally and > 4 post-ACTH), while 3 had bilateral source during AVS study. In the 3 patients undergoing unilateral adrenalectomy for lateralized source and contralateral suppression and adequate follow-up data, cure of PA was achieved at mean 58 months postoperatively.
CONCLUSION
AVS using dexamethasone premedication is safe and accurate for diagnosing the source of aldosterone excess in patients with PA and ICM allergy.
PubMed: 35795806
DOI: 10.1210/jendso/bvac093 -
The Journal of Biological Chemistry Aug 2009Elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein...
Elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca(2+) ionophore, 4-bromo-A23187 (0.5 mum): 1) inhibited NHE3 V(max) activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca(2+)](i) by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca(2+)](i) inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca(2+)](i); 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca(2+).
Topics: Amino Acid Motifs; Caco-2 Cells; Calcium; Carrier Proteins; Cell Line; Cell Membrane; Down-Regulation; Humans; Membrane Proteins; Protein Binding; Protein Transport; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers
PubMed: 19535329
DOI: 10.1074/jbc.M109.012641 -
Cellular and Molecular Gastroenterology... 2023Fiber-rich foods promote health, but mechanisms by which they do so remain poorly defined. Screening fiber types, in mice, revealed psyllium had unique ability to...
BACKGROUND & AIMS
Fiber-rich foods promote health, but mechanisms by which they do so remain poorly defined. Screening fiber types, in mice, revealed psyllium had unique ability to ameliorate 2 chronic inflammatory states, namely, metabolic syndrome and colitis. We sought to determine the mechanism of action of the latter.
METHODS
Mice were fed grain-based chow, which is naturally rich in fiber or compositionally defined diets enriched with semi-purified fibers. Mice were studied basally and in models of chemical-induced and T-cell transfer colitis.
RESULTS
Relative to all diets tested, mice consuming psyllium-enriched compositionally defined diets were markedly protected against both dextran sulfate sodium- and T-cell transfer-induced colitis, as revealed by clinical-type, histopathologic, morphologic, and immunologic parameters. Such protection associated with stark basal changes in the gut microbiome but was independent of fermentation and, moreover, maintained in mice harboring a minimal microbiota (ie, Altered Schaedler Flora). Transcriptomic analysis revealed psyllium induced expression of genes mediating bile acids (BA) secretion, suggesting that psyllium's known ability to bind BA might contribute to its ability to prevent colitis. As expected, psyllium resulted in elevated level of fecal BA, reflecting their removal from enterohepatic circulation but, in stark contrast to the BA sequestrant cholestyramine, increased serum BA levels. Moreover, the use of BA mimetics that activate the farnesoid X receptor (FXR), as well as the use of FXR-knockout mice, suggested that activation of FXR plays a central role in psyllium's protection against colitis.
CONCLUSIONS
Psyllium protects against colitis via altering BA metabolism resulting in activation of FXR, which suppresses pro-inflammatory signaling.
Topics: Mice; Animals; Psyllium; Bile Acids and Salts; Health Promotion; Colitis; Inflammation; Mice, Knockout
PubMed: 36828279
DOI: 10.1016/j.jcmgh.2023.02.007