Biomedical subjects

Ferenc Erdodi

Publications and source records attributed to Ferenc Erdodi.

12 recordsLinked to original sources

PubMed2006

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells.

AIM: To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in inflammatory bowel disease (IBD). METHODS: We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10(-4) mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS: In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18%+/-16% vs 23%+/-15% P<0.01). UC or CD serum caused a significant increase in iNOS compared to control (UC: 300%+/-21%; CD: 275%+/-27% vs 108%+/-14%, P<0.01). Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION: IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced anti-inflammatory protection of endothelial cells.

Adolescent↗

PubMed2005

Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: inhibitory effects and occurrence in A7r5 cells.

Major sites for Rho-kinase on the myosin phosphatase target subunit (MYPT1) are Thr695 and Thr850. Phosphorylation of Thr695 inhibits phosphatase activity but the role of phosphorylation at Thr850 is not clear and is evaluated here. Phosphorylation of both Thr695 and Thr850 by Rho-kinase inhibited activity of the type 1 phosphatase catalytic subunit. Rates of phosphorylation of the two sites were similar and efficacy of inhibition following phosphorylation was equivalent for each site. Phosphorylation of each site on MYPT1 was detected in A7r5 cells, but Thr850 was preferred by Rho-kinase and Thr695 was phosphorylated by an unidentified kinase(s).

Animals↗

PubMed2005

Localization of myosin phosphatase target subunit and its mutants.

Transient transfection of NIH3T3 cells with various constructs of myosin phosphatase target subunit (MYPT1) and GFP showed distinct cellular localizations. Constructs containing the N-terminal nuclear localization signals (NLS), i.e. full-length MYPT1 and N-terminal MYPT1 fragments, were concentrated in the nucleus. Full-length chicken and human MYPT1-GFP showed discrete nuclear foci. Deletion of the N-terminal NLS or use of central or C-terminal MYPT1 fragments did not show unique nuclear distributions (C-terminal NLS are present). Transient transfection of NIH3T3 cells (in the presence of serum) with full-length MYPT1-GFP caused a marked decrease in number of attached cells, an apparent block in the cell cycle prior to M phase and signs of increased apoptosis. Under conditions of serum starvation the unique nuclear localization of MYPT1-GFP was not found and there was no marked decrease in the number of attached cells (after 48 h). Stable transfection of HEK 293 cells with GFP-MYPT1 was obtained. MYPT1 and its N-terminal mutants bound to retinoblastoma protein (Rb), raising the possibility that Rb is implicated in the effects caused by overexpression of MYPT1.

Animals↗

PubMed2005

Okadaic acid induces phosphorylation and translocation of myosin phosphatase target subunit 1 influencing myosin phosphorylation, stress fiber assembly and cell migration in HepG2 cells.

It was determined that the myosin phosphatase (MP) activity and content of myosin phosphatase target subunit 1 (MYPT1) were correlated in subcellular fractions of human hepatocarcinoma (HepG2) cells. In control cells MYPT1 was localized in the cytoplasm and in the nucleus, as determined by confocal microscopy. Treatment of HepG2 cells with 50 nM okadaic acid (OA), a cell-permeable phosphatase inhibitor, induced several changes: 1) a marked redistribution of MYPT1 to the plasma membrane associated with an increased level of phosphorylation of MYPT1 at Thr695. Both effects showed only a slight influence with the Rho-kinase inhibitor, Y-27632; 2) an increase in phosphorylation of MYPT1 at Thr850 associated with its accumulation in the perinuclear region and nucleus. These effects were markedly reduced by Y-27632; 3) an increased phosphorylation of the 20 kDa myosin II light chain at Ser19 associated with an increased location of myosin II at the cell center. These effects were partially counteracted by Y-27632; 4) an increase in stress fiber formation and a decrease in cell migration, both OA-induced effects were blocked by Y-27632. In HepG2 lysates, OA (5-100 nM) did not affect MP activity but inhibited PP2A activity. These results indicate that OA induces differential phosphorylation and translocation of MYPT1, dependent on PP2A and, to varying extents, on ROK. These changes are associated with an increased level of myosin II phosphorylation and attenuation of hepatic cell migration.

Amides↗

PubMed2005

Changes in the expression and distribution of the inducible and endothelial nitric oxide synthase in mucosal biopsy specimens of inflammatory bowel disease.

OBJECTIVE: The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel disease (IBD) is controversial. The aim of this study was to investigate the expression and localization of nitric oxide synthase isoforms (iNOS, eNOS) in IBD colonic mucosa. MATERIAL AND METHODS: Forty-four patients with IBD (24 ulcerative colitis (UC), 20 Crohn's disease (CD) and 16 controls) were investigated by colonoscopy. iNOS and eNOS in tissue sections was demonstrated by histochemistry (NADPH-diaphorase reaction) and immunohistochemistry. Cell type analysis and quantitative assessment of the iNOS immunoreactive (IR) cells and densitometry of iNOS in immunoblots were also performed. RESULTS: iNOS-IR cells were significantly numerous in inflamed mucosa of UC (64+/-4 cells/mm2) than in CD (4+/-2 cells/mm2). iNOS-IR/CD15-IR cells showed significant elevation in inflamed (i) versus uninflamed (u) UC mucosa (UCu 8+/-3%, UCi 85+/-10%) In CD, the percentage of iNOS-IR/CD68-IR cells was lower in inflamed sites (CDu 23+/-8%, CDi 4+/-3%). Immunoblot of biopsies revealed significant elevation of iNOS in active UC compared with uninflamed sites, whereas in CD no significant changes were detected. Differences were observed in eNOS and endothelial marker CD31 immunoreactivity. In patients with UC and in controls the ratios of eNOS/CD31-IR vessels were 82.3% and 92.0% respectively, whereas in CD the ratio was 8.3% with a concomitantly significant increase of CD31-IR vessels. The distribution and morphological characteristics of the NOS-IR inflammatory cells and endothelia were similar to those showing NADPH-diaphorase reactivity. CONCLUSIONS: Differences observed in the expression and distribution of NOS isoforms in immune and endothelial cells may contribute to better understanding of the structural and physiological changes in UC and CD.

Adult↗

PubMed2004

Localization of myosin phosphatase target subunit 1 in rat brain and in primary cultures of neuronal cells.

Myosin phosphatase (PP1M) is composed of the delta isoform of the PP1 catalytic subunit (PP1cdelta), the myosin phosphatase target subunit (MYPT), and a 20 kDa subunit. Western blots detected higher amounts of the MYPT1 isoform compared to MYPT2 in whole brain extracts. The localization of MYPT1 was studied in rat brain and in primary cell cultures of neurons using specific antibodies. Analysis of lysates of brain regions for MYPT1 and PP1M by Western blots using anti-MYPT1 antibodies and by phosphatase assays with myosin as substrate suggested a ubiquitous distribution. Immunohistochemistry of tissue sections revealed that MYPT1 was distributed in all areas of the brain, with staining observed in many different cell types. Depending on the method used for fixation, the MYPT1 appeared with varying intensity in nuclei, in nucleoli, and in the cytoplasm. In primary hippocampal cultures, MYPT1 was identified by confocal microscopy in the cytoplasm and in the nucleus, whereas a predominantly cytoplasmic localization was found in cochlear nucleus cells. In cultured cells, MYPT1 and PP1cdelta colocalized with synaptophysin. PP1M activity was high in synaptosomes isolated from the cerebral cortex, but was relatively low in the postsynaptic densities. The interaction of MYPT1 with synaptophysin and with known partners (Rho-kinase, PP1cdelta) in brain extracts was shown by immunoprecipitation with anti-MYPT1. Pull-down assays from synaptosomes, using GST-MYPT1, also confirmed these interactions. In conclusion, the widespread cellular and subcellular localization of MYPT1 implies that PP1M may play an important role in the dephosphorylation of key regulatory proteins in neuronal cells.

Animals↗

PubMed2004

Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides.

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

Animals↗

PubMed2004

Myosin phosphatase: structure, regulation and function.

Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PPlc; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility.

Animals↗

PubMed2003

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases.

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.

Animals↗

PubMed2003

Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells.

C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, delta isoform (PP1c delta). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PP1c delta was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PP1c, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PP1c delta, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3-5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.

Animals↗

PubMed2002

Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase.

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695)-->Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

Amino Acid Sequence↗

PubMed2002

Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.

The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5'-[gamma-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was not affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1(667-1004)). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.

Adenosine Triphosphate↗