The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha(2)beta(1)

Background: Activation of the platelet integrin alpha(2)beta(1) is closely regulated due to the high thrombogenicity of its ligand. As a beta(1) interacting kinase, ILK represents a candidate intracellular regulator of alpha(2)beta(1) in human platelets. Objectives We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha(2)beta(1) activation in HEL cells, a megakaryocytic cell line. Methods: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta(1)-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha(2)beta(1) function assessed by overexpression studies in HEL cells. Results: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta(1)-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha(2)beta(1)-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha(2)beta(1). Conclusions: Our findings that ILK regulates alpha(2)beta(1) in HEL cells, is activated in platelets and associates with beta(1)-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.

Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?

In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification of a serine protease involved with the regulation of adrenal growth

The adrenal cortex is a dynamic organ in which the cells of the outer cortex continually divide. It is well known that this cellular proliferation is dependent on constant stimulation from peptides derived from the ACTH precursor pro-opiomelanocortin (POMC) because disruption of pituitary corticotroph function results in rapid atrophy of the gland. Previous results from our laboratory have suggested that the adrenal mitogen is a fragment derived from the N-terminal of POMC not containing the gamma-MSH sequence. Because such a peptide is not generated during processing of POMC in the pituitary, we proposed that the mitogen is generated from circulating pro-gamma-MSH by an adrenal protease. Using degenerate oligonucleotides, we identified a secreted serine protease expressed by the adrenal gland that we named adrenal secretory protease (ASP). In the adrenal cortex, expression of ASP is limited to the outer zona glomerulosa/fasciculata, the region where cortical cells are believed to be derived, and is significantly up-regulated during compensatory growth. Y1 adrenocortical cells transfected with a vector expressing an antisense RNA (and thus having reduced levels of endogenous ASP) were found to grow slower than sense controls while also losing their ability to utilize exogenous pro-gamma-MSH in the media supporting a role for ASP in adrenal growth. Digestion of an N-POMC peptide substrate encompassing the residues around the dibasic cleavage site at positions 49/50 with affinity-purified ASP showed cleavage not to occur at the dibasic site but two residues downstream leading us to propose the identity of the adrenal mitogen to be N-POMC (1-52).

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Ownership rights in performance-based contracting: A case study of English law

Clients and contractors need to be aware of the project’s legal environment because the viability of a procurement strategy can be vitiated by legal rules. This is particularly true regarding Performance-Based Contracting (PBC) whose viability may be threatened by rules of property law: while the PBC concept does not require that the contractor transfers the ownership in the building materials used to the client, the rules of property law often lead to an automatic transfer of ownership. But does the legal environment really render PBC unfeasible? In particular, is PBC unfeasible because contractors lose their materials as assets? These questions need to be answered with respect to the applicable property law. As a case study, English property law has been chosen. Under English law, the rule which governs the automatic transfer of ownership is called quicquid plantatur solo, solo credit (whatever is fixed to the soil belongs to the soil). An analysis of this rule reveals that not all materials which are affixed to land become part of the land. This fate only occurs in relation to materials which have been affixed with the intention of permanently improving the land. Five fictitious PBC cases have been considered in terms of the legal status of the materials involved, and several subsequent legal questions have been addressed. The results suggest that English law does actually threaten the feasibility of PBC in some cases. However, it is also shown that the law provides means to circumvent the unwanted results which flow from the rules of property law. In particular, contractors who are interested in keeping their materials as assets can insist on agreeing a property right in the client’s land, i.e. a contractor’s lien. Therefore, the outcome is that English property law does not render the implementation of the PBC concept unfeasible. At a broader level, the results contribute to the theoretical framework of PBC as an increasingly used procurement strategy.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.

Regulation of the neural circuitry of emotion by compassion meditation: effects of meditative expertise

Recent brain imaging studies using functional magnetic resonance imaging (fMRI) have implicated insula and anterior cingulate cortices in the empathic response to another's pain. However, virtually nothing is known about the impact of the voluntary generation of compassion on this network. To investigate these questions we assessed brain activity using fMRI while novice and expert meditation practitioners generated a loving-kindness-compassion meditation state. To probe affective reactivity, we presented emotional and neutral sounds during the meditation and comparison periods. Our main hypothesis was that the concern for others cultivated during this form of meditation enhances affective processing, in particular in response to sounds of distress, and that this response to emotional sounds is modulated by the degree of meditation training. The presentation of the emotional sounds was associated with increased pupil diameter and activation of limbic regions (insula and cingulate cortices) during meditation (versus rest). During meditation, activation in insula was greater during presentation of negative sounds than positive or neutral sounds in expert than it was in novice meditators. The strength of activation in insula was also associated with self-reported intensity of the meditation for both groups. These results support the role of the limbic circuitry in emotion sharing. The comparison between meditation vs. rest states between experts and novices also showed increased activation in amygdala, right temporo-parietal junction (TPJ), and right posterior superior temporal sulcus (pSTS) in response to all sounds, suggesting, greater detection of the emotional sounds, and enhanced mentation in response to emotional human vocalizations for experts than novices during meditation. Together these data indicate that the mental expertise to cultivate positive emotion alters the activation of circuitries previously linked to empathy and theory of mind in response to emotional stimuli.

Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Growth of melanocytic cells is associated with down-regulation of protein kinase C α,δ and ε isoforms: Possible role of diacylglycerol

Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.

Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased ( approximately 9-fold; 15-30 min) with later increases in expression of Klf4 and Klf6 ( approximately 5-fold; 30-60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1-2 h ( approximately 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1beta or tumor necrosis factor alpha downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli.