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Myosin Light Chain Kinase (myosin + light_chain_kinase)
Selected AbstractsMyosin Light Chain Kinase as a Multifunctional Regulatory Protein of Smooth Muscle ContractionIUBMB LIFE, Issue 6 2001Ying Gao Abstract Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20-kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well-established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin-binding activity at its N terminal and myosin-binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin-myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction. [source] Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytesCYTOSKELETON, Issue 7 2006T. V. Dudnakova Abstract Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the abovementioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Myosin Light Chain Kinase as a Multifunctional Regulatory Protein of Smooth Muscle ContractionIUBMB LIFE, Issue 6 2001Ying Gao Abstract Myosin light chain kinase (MLCK) is a regulatory protein for smooth muscle contraction, which acts by phosphorylating 20-kDa myosin light chain (MLC20) to activate the myosin ATPase activity. Although this mode of action is well-established, there are numerous reports of smooth muscle contraction that is not associated with MLC20 phosphorylation. The kinase activity for the phosphorylation is localized at the central part of MLCK, which is also furnished with actin-binding activity at its N terminal and myosin-binding activity at its C terminal. This article overviews as to how such multifunctional properties of MLCK modify the actin-myosin interaction and presents our observations that the phosphorylation is not obligatory in induction of smooth muscle contraction. [source] In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesisCYTOSKELETON, Issue 2 2008Ryota Uehara Abstract Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Causal mapping as a tool to mechanistically interpret phenomena in cell motility: Application to cortical oscillations in spreading cellsCYTOSKELETON, Issue 9 2006Gabriel E. Weinreb Abstract Biological processes that occur at the cellular level and consist of large numbers of interacting elements are highly nonlinear and generally involve multiple time and spatial scales. The quantitative description of these complex systems is of great importance but presents large challenges. We outline a new systems biology approach, causal mapping (CMAP), which is a coarse-grained biological network tool that permits description of causal interactions between the elements of the network and overall system dynamics. On one hand, the CMAP is an intermediate between experiments and physical modeling, describing major requisite elements, their interactions and paths of causality propagation. On the other hand, the CMAP is an independent tool to explore the hierarchical organization of cell and the role of uncertainties in the system. It appears to be a promising easy-to-use technique for cell biologists to systematically probe verbally formulated qualitative hypotheses. We apply the CMAP to study the phenomenon of contractility oscillations in spreading cells in which microtubules have been depolymerized. The precise mechanism by which these oscillations are governed by a complex mechano-chemical system is not known but the data observed in experiments can be described by a CMAP. The CMAP suggests that the source of the oscillations results from the opposing effects of Rho activation leading to a decreased level of myosin light chain phosphatase and a cyclic calcium influx caused by increased membrane tension and leading to a periodically enhanced activation of myosin light chain kinase. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Myosin-II negatively regulates minor process extension and the temporal development of neuronal polarityDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2009K.M. Kollins Abstract The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Protein phosphorylation pathways involved during lipopolysaccharide-induced expression of CD14 in mouse bone marrow granulocytesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2000Thierry Pedron Abstract Lipopolysaccharide (LPS) of Gram-negative bacteria interacts with a CD14-independent receptor of mouse bone marrow granulocytes (BMC), and triggers in these cells the expression of CD14, an inducible type of LPS receptor (iLpsR). This particular response of BMC to LPS required the activation of protein tyrosine kinase and p38 MAP kinase. The inhibition of the LPS effect by the MEK inhibitor PD-98059 suggested that the ERK pathway was also involved. Unexpectedly, protein kinase C, myosin light chain kinase, cAMP-, cGMP-, and Ca2+/calmodulin-dependent kinases, as well as ecto-protein kinases, were not required for iLpsR expression. However, other yet unidentified serine/threonine protein kinase(s) were implied since the BMC response to LPS was markedly reduced after exposure to three inhibitors of such kinases (K-252a, H-7, and KT-5823). The atypical kinase requirements observed in this study may be due either to a novel signaling LPS receptor complex present in BMC, or to the particular events involved in CD14 biosynthesis. [source] A common cortactin gene variation confers differential susceptibility to severe asthmaGENETIC EPIDEMIOLOGY, Issue 8 2008Shwu-Fan Ma Abstract Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20,2.43; p=0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40,6.13, p=0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants. Genet. Epidemiol. 2008. © 2008 Wiley-Liss, Inc. [source] A variant of the myosin light chain kinase gene is associated with severe asthma in African AmericansGENETIC EPIDEMIOLOGY, Issue 4 2007Carlos Flores Abstract Asthma is a complex phenotype influenced by environmental and genetic factors for which severe irreversible structural airway alterations are more frequently observed in African Americans. In addition to a multitude of factors contributing to its pathobiology, increased amounts of myosin light chain kinase (MLCK), the central regulator of cellular contraction, have been found in airway smooth muscle from asthmatics. The gene encoding MLCK (MYLK) is located in 3q21.1, a region noted by a number of genome-wide studies to show linkage with asthma and asthma-related phenotypes. We studied 17 MYLK genetic variants in European and African Americans with asthma and severe asthma and identified a single non-synonymous polymorphism (Pro147Ser) that was almost entirely restricted to African populations and which was associated with severe asthma in African Americans. These results remained highly significant after adjusting for proportions of ancestry estimated using 30 unlinked microsatellites (adjusted odds ratio: 1.76 [95% confidence interval, CI: 1.17,2.65], p = 0.005). Since all common HapMap polymorphisms in ,500,kb contiguous regions have low-to-moderate linkage disequilibrium with Pro147Ser, we speculate that this polymorphism is causally related to the severe asthma phenotype in African Americans. The association of this polymorphism, located in the N-terminal region of the non-muscle MLCK isoform, emphasizes the potential importance of the vascular endothelium, a tissue in which MLCK is centrally involved in multiple aspects of the inflammatory response, in the pathogenesis of severe asthma. This finding also offers a possible genetic explanation for some of the more severe asthma phenotype observed in African American asthmatics. Genet Epidemiol 2007. © 2007 Wiley-Liss, Inc. [source] An MLCK-dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK-p70S6K pathway,HEPATOLOGY, Issue 1 2006Anne Bessard We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant-negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2-dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK-dependent window in late G1 phase of regenerating liver after two-thirds partial hepatectomy. In conclusion, our results underline an MLCK-dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation. (HEPATOLOGY 2006;44:152,163.) [source] Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cellJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2007Vladimír Pucovský Abstract This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle ,-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmis-sion electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries. [source] Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2010T. M. GETZ Summary. Background:, Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK). Objective:, The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets. Results:, Induction of 2MeSADP-induced shape change occurs within 5 s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G12/13 produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160ROCK inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca2+ -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G12/13 -regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch. [source] The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cellsPROTEIN SCIENCE, Issue 3 2010Hao Huang Abstract Soybean calmodulin isoform 4 (sCaM4) is a plant calcium-binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo-) form of sCaM4 possesses a half unfolded structure, with the N-terminal domain unfolded and the C-terminal domain folded. This result was unexpected as the apo-forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high concentrations in cells (0.5,2 mM), we suggest that Mg2+ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg2+ the initially unfolded N-terminal domain of sCaM4 folds into an ,-helix-rich structure, similar to the Ca2+ form. We have used the NMR backbone residual dipolar coupling restraints 1DNH, 1DC,H,, and 1DC,C, to determine the solution structure of the N-terminal domain of Mg2+ -sCaM4 (Mg2+ -sCaM4-NT). Compared with the known structure of Ca2+ -sCaM4, the structure of the Mg2+ -sCaM4-NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg2+ -sCaM4 and CaM-binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg2+ -sCaM4 does not bind to Ca2+ -CaM target peptides and therefore is functionally similar to apo-mCaM. The Mg2+ - and apo-structures of the sCaM4-NT provide unique insights into the structure and function of some plant calmodulins in resting cells. [source] Role of sarcoplasmic reticulum in control of membrane potential and nitrergic response in opossum lower esophageal sphincterBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2003Yong Zhang We previously demonstrated that a balance of Ca2+ -activated Cl, current (ICl(Ca)) and K+ current activity sets the resting membrane potential of opossum lower esophageal sphincter (LES) circular smooth muscle at ,,41 mV, which leads to continuous spike-like action potentials and the generation of basal tone. Ionic mechanisms underlying this basal ICl(Ca) activity and its nitrergic regulation remain unclear. Recent studies suggest that spontaneous Ca2+ release from sarcoplasmic reticulum (SR) and myosin light chain kinase (MLCK) play important roles. The current study investigated this possibility. Conventional intracellular recordings were performed on circular smooth muscle of opossum LES. Nerve responses were evoked by electrical square wave pulses of 0.5 ms duration at 20 Hz. In the presence of nifedipine (1 ,M), substance P (1 ,M), atropine (3 ,M) and guanethidine (3 ,M), intracellular recordings demonstrated a resting membrane potential (MP) of ,38.1±0.7 mV (n=25) with spontaneous membrane potential fluctuations (MPfs) of 1,3 mV. Four pulses of nerve stimulation induced slow inhibitory junction potentials (sIJPs) with an amplitude of 6.1±0.3 mV and a half-amplitude duration of 1926±147 ms (n=25). 1H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific guanylyl cyclase inhibitor, abolished sIJPs, but had no effects on MPfs. Caffeine, a ryanodine receptor agonist, hyperpolarized MP and abolished sIJPs and MPfs. Ryanodine (20 ,M) inhibited the sIJP and induced biphasic effects on MP, an initial small hyperpolarization followed by a large depolarization. sIJPs and MPfs were also inhibited by cyclopiazonic acid, an SR Ca2+ ATPase inhibitor. Specific ICl(Ca) and MLCK inhibitors hyperpolarized the MP and inhibited MPfs and sIJPs. These data suggest that (1) spontaneous release of Ca2+ from the SR activates ICl(Ca), which in turn contributes to resting membrane potential; (2) MLCK is involved in activation of ICl(Ca); (3) inhibition of ICl(Ca) is likely to underlie sIJPs induced by nitrergic innervation. British Journal of Pharmacology (2003) 140, 1097,1107. doi:10.1038/sj.bjp.0705537 [source] Mechanisms of 17 ,-oestradiol induced vasodilatation in isolated pressurized rat small arteriesBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2000Linda Shaw The influence of 17 ,-oestradiol on pressurized isolated rat mesenteric and coronary small arteries was investigated. 17 ,-oestradiol caused rapid (t1.0<5 mins) concentration-dependent relaxations of pre-contracted pressurized (50 mmHg) isolated rat mesenteric and coronary arteries. Similar responses were observed in both vessel types. Significant relaxations were only observed at concentrations exceeding 3 ,M. The vasodilatory responses in both types of artery were unaffected by 10 ,ML -nitro arginine (L -NNA) alone or in the presence of 10 ,M indomethacin, inhibitors of nitric oxide and prostaglandin synthesis respectively. They were also unaffected by the pre-contracting agent used i.e. high K+ or U46619 (a thromboxane analogue). Neither the oestrogen receptor antagonist ICI 182,780 (10 ,M) nor the protein synthesis inhibitor cycloheximide (100 ,M) had any effect on the responses of mesenteric arteries to 17 ,-oestradiol. 17 ,-oestradiol had only a minor effect on mesenteric arterial diameter over a concentration range similar to the effective vasodilatory range for 17 ,-oestradiol. Membrane impermeant 17 ,-oestradiol conjugated to bovine serum albumin (,-oestradiol-17hemisuccinate-BSA) (E-H-BSA) resulted in a vasodilatation of pressurized arteries. Wortmannin, an inhibitor of myosin light chain kinase, near maximally relaxed pressurized mesenteric arteries although the time course for the response was significantly slower than that for 17 ,-oestradiol. These results taken together suggest that the acute effects of 17 ,-oestradiol on isolated pressurized arterial tone may be due to effects directly on the vascular smooth muscle via non-genomic mechanisms that involve a stereospecific interaction at the plasma membrane. British Journal of Pharmacology (2000) 129, 555,565; doi:10.1038/sj.bjp.0703084 [source] A ROLE FOR RHO-KINASE IN Ca2+ -INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL-12,13-DIBUTYRATECLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 3 2009Inji Baek SUMMARY 1Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca2+ -depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca2+ -independent contractions induced by PDBu in vascular smooth muscles. 2In Ca2+ -free solution, 0.1 and 1 µmol/L PDBu induced contraction and myosin light chain (MLC20) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 µmol/L), an inhibitor of Rho-kinase, but not ML7 (10 µmol/L), an inhibitor of myosin light chain kinase, inhibited Ca2+ -independent contractions induced by PDBu. 3In Ca2+ -free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 µmol/L Ro31-8220, an inhibitor of PKC. 4In Ca2+ -free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5In conclusion, RhoA/Rho-kinase plays an important role in Ca2+ -independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca2+ -independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17. [source] BMP-2 and FGF-2 Synergistically Facilitate Adoption of a Cardiac Phenotype in Somatic Bone Marrow c-kit+/Sca-1+ Stem CellsCLINICAL AND TRANSLATIONAL SCIENCE, Issue 2 2008Brent R. DeGeorge, Jr. B.S. Abstract The aim of this study was to explore the effect of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2), paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI),on murine bone marrow stem cell (mBMSC) differentiation in an ex vivo cardiac microenvironment. For this purpose, green fluorescent protein (GFP) expressing hematopoietic lineage negative (lin-) c-kit ligand (c-kit) and stem cell antigen-1 (Sca-1) positive (GFP-lin-/c-kit+/sca+) mBMSC were co-cultured with neonatal rat ventricular cardiomyocytes (NVCMs). GFP+ mBMSC significantly induced the expression of BMP-2 and FGF-2 in NVCMs, and approximately 4% GFP+ mBMSCs could be recovered from the co-culture at day 10. The addition of BMP-2 in concert with FGF-2 significantly enhanced the amount of integrated GFP+ mBMSCs by 5-fold (,20%), whereas the addition of anti-BMP-2 and/or anti-FGF-2 antibodies completely abolished this effect. An analysis of calcium cycling revealed robust calcium transients in GFP+ mBMSCs treated with BMP-2/FGF-2 compared to untreated co-cultures. BMP-2 and FGF-2 addition led to a significant induction of early (NK2 transcription factor related, locus 5; Nkx2.5, GATA binding protein 4; GATA-4) and late (myosin light chain kinase [MLC-2v], connexin 43 [Cx43]) cardiac marker mRNA expression in mBMSCs following co-culture. In addition, re-cultured fluorescence-activated cell sorting (FACS)-purified BMP-2/FGF-2-treated mBMSCs revealed robust calcium transients in response to electrical field stimulation which were inhibited by the L-type calcium channel (LTCC) inhibitor, nifedipine, and displayed caffeine-sensitive intracellular calcium stores. In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. This study provides the first evidence that mBMSCs with long-term self-renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross-talk within an appropriate cardiac microenvironment. [source] Expression of muscle-related genes and two MyoD genes during amphioxus notochord developmentEVOLUTION AND DEVELOPMENT, Issue 5 2003Aki Urano Summary The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expre-ssion patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropo-myosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), Amphi-nTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expres-sed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle. [source] | |||||||||||||