Intracellular Biochemical Signals Research Articles

G-Protein coupled membrane receptors may serve as mechanosensors for fluid shear stress in neutrophils

This chapter reviews the mechanical properties of prominent matrix proteins and the nature of cell attachments to the matrix. The chapter discusses the process of interaction of cell-matrix attachments with the cytoskeleton to regulate stretch sensitivity of mechanosensitive channels, and analyzes the process that matrix proteins may interact with mechanosensors to affect mechanotransduction. Mechanosensation involves the ability of cells to detect forces and explore the topography of the extracellular matrix. This information about the physical environment is then translated into intracellular biochemical signals, a process known as “mechanotransduction” that leads to the generation of cellular responses (mechanoresponses) that may impact the structure and function of the extracellular matrix itself. Target cell adaptation and desensitization of mechanosensitive channels to constant or repeated mechanical stimulation in connective tissue fibroblasts has been reported and may prevent “information overload” including overt Ca2+ induced cell death. Cellular adaptations to applied mechanical forces, including the regulation of mechanosensitive channel function, is important for optimizing cellular responses to fluctuations in the physical environment of cells.

Stepwise mechanical stretching inhibits chondrogenesis through cell–matrix adhesion mediated by integrins in embryonic rat limb-bud mesenchymal cells

Biomechanical forces are major epigenetic factors that determine the form and differentiation of skeletal tissues, and may be transduced through cell adhesion to the intracellular biochemical signaling pathway. To test the hypothesis that stepwise stretching is translated to molecular signals during early chondrogenesis, we developed a culture system to study the proliferation and differentiation of chondrocytes. Rat embryonic day-12 limb buds were microdissected and dissociated into cells, which were then micromass cultured on a silicone membrane and maintained for up to 7 days. Stepwise-increased stretching was applied to the silicone membrane, which exerted shearing stress on the cultures on day 4 after the initiation of chondrogenesis. Under stretched conditions, type II collagen expression was significantly inhibited by 44% on day 1 and by 67% on day 2, and this difference in type II collagen reached 80% after 3 days of culture. Accumulation of type II collagen protein and the size of the chondrogenic nodules had decreased by 50% on day 3. On the other hand, expression of the non-chondrogenic marker fibronectin was significantly up-regulated by 1.8-fold on day 3, while the up-regulation of type I collagen was minimal, even by day 3. The down-regulation in the expression of chondrogenic markers was completely recovered when cell–extracellular matrix attachment was inhibited by Gly–Arg–Gly–Asp–Ser–Pro–Lys peptide or by the application of blocking antibodies for α2, α5 or β1 integrins. We conclude that shearing stress generated by stepwise stretching inhibits chondrogenesis through integrins, and propose that signal transduction from biomechanical stimuli may be mediated by cell–extracellular matrix adhesion.

Directional dependence of osteoblastic calcium response to mechanical stimuli.

In adaptive bone remodeling, mechanical signals such as stress/strain caused by loading/deformation are believed to play important roles as regulators of the process in which osteoclastic resorption and osteoblastic formation are coordinated under a local mechanical environment. The mechanism by which cells sense and transduce mechanical signals to the intracellular biochemical signaling cascade is still unclear, however to address this issue, the present study investigated the characteristic response of a single osteoblastic cell, MC3T3-E1, to a well-defined mechanical stimulus and the involvement of the cytoskeletal actin fiber structure in the mechanotransduction pathway. First, by mechanically perturbing to a single cell using a microneedle, a change in the intracellular calcium ion concentration [Ca(2+)](i) was observed as a primal signaling response to a mechanical stimulus, and the threshold value of the perturbation as the mechanical stimulus was evaluated quantitatively. Second, to study directional dependence of the response to the mechanical stimulus, the effect of actin fiber orientation on the threshold value of the calcium response was investigated at various magnitudes and directions of the stimulus. It was found that the osteoblastic response to the perturbation exhibited a directional dependence. That is, the sensitivity of osteoblastic cells to a mechanical stimulus depends on the angle of the applied deformation with respect to the cytoskeletal actin fiber orientation. This finding is phenomenological evidence that cytoskeletal actin fiber structures are involved in the mechanotransduction mechanism, which may be related to cell polarization behaviors such as cellular alignment caused by mechanical stimulation.

FINDING CYCLES IN SYNCHRONOUS BOOLEAN NETWORKS WITH APPLICATIONS TO BIOCHEMICAL SYSTEMS

This paper is an analytical study of Boolean networks. The motivation is our desire to understand the large, complicated and interconnected pathways which comprise intracellular biochemical signal transduction networks. The simplest possible conceptual model that mimics signal transduction with sigmoidal kinetics is the n-node Boolean network each of whose elements or nodes has the value 0 (off) or 1 (on) at any given time T = 0, 1, 2, …. A Boolean network has 2nstates all of which are either on periodic cycles (including fixed points) or transients leading to cycles. Thus one understands a Boolean network by determining the number and length of its cycles. The problem one must circumvent is the large number of states (2n) since the networks we are interested in have 100 or more elements. Thus we concentrate on developing size n methods rather than the impossible task of enumerating all 2nstates. This is done as follows: the dynamics of the network can be described by n polynomial equations which describe the logical function which determines the interaction at each node. Iterating the equations one step at a time finds all fixed points, period two cycles, period three cycles, etc. This is a general method that can be used to determine the fixed points and moderately large periodic cycles of any size network, but it is not useful in finding the largest cycles in a large network. However, we also show that the network equations can often be reduced to scalar form, which makes the cycle structure much more transparent. The scalar equations method is a true "size n" method and several examples (including nontrivial biochemical systems) are examined.

A novel pleiotropic effect of statins: prevention of cardiac hypertrophy by cholesterol-independent mechanisms

Cardiac hypertrophy is an initial physiological adaptive response by the heart to pressure overload. However, if pressure overload persists, frequently, the heart decompensates and develops 'pathophysiological' hypertrophy. This leads to increased mortality and morbidity and is an independent risk factor for heart failure. Because cardiac myocytes convert this pressure overload into intracellular biochemical signals, blocking this critical signaling pathway may be an important therapeutic target to prevent cardiac hypertrophy. Small GTP-binding proteins, in particular Rac1, have been suggested to play a key role in the development of cardiac hypertrophy. Recently, 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, also called statins, have been shown to inhibit cardiac hypertrophy independent of their cholesterol lowering property. Statins block the isoprenylation and activation of members of the Rho family, such as RhoA and Rac1. Rac1 also regulates NADPH oxidase, which is a major source of reactive oxygen species (ROS) in cardiovascular cells. Growing evidence suggests that ROS may be involved in the process of cardiac hypertrophy and recent research has shown that statins attenuate oxidative stress through inhibition of Rac1. Overall, these pleiotropic effects of statins will give new insights into the process of cardiac hypertrophy.

Molecular mechanism of cardiac hypertrophy and development.

Congestive heart failure is a major issues for cardiologists and to fully understand heart failure, it is important to understand the mechanism of the development of cardiac hypertrophy. Hemodynamic overload, namely mechanical stress, is a major cause of cardiac hypertrophy and to dissect the signaling pathways from mechanical stress to cardiac hypertrophy, an in-vitro device by which mechanical stress can be imposed on cardiac myocytes of neonatal rats cultured in serum-free conditions has been developed. Passively stretching cardiac myocytes cultured on silicone membranes induced various hypertrophic responses, such as activation of the phosphorylation cascades of many protein kinases, expression of specific genes and an increase in protein synthesis. During this process, secretion and production of vasoactive peptides, such as angiotensin II and endothelin-1, were increased and they played critical roles in the induction of these hypertrophic responses. Candidates for the 'mechanoreceptor' that receives the mechanical stress and converts it into intracellular biochemical signals have been recently demonstrated. Gene therapy and cell transplantation are hopeful strategies for the treatment of heart failure and require an understanding of how normal cardiac myocytes are differentiated. A key gene that plays a critical role in cardiac development has been isolated. The cardiac homeobox-containing gene Csx is expressed in the heart and the heart progenitor cells from the very early developmental stage, and targeted disruption of the murine Csx results in embryonic lethality because of the abnormal looping morphogenesis of the primary heart tube. With a cardiac zinc finger protein GATA4, Csx induces cardiomyocyte differentiation of teratocarcinoma cells as well as upregulation of cardiac genes. Mutations of human CSX cause various congenital heart diseases including atrial septal defect, ventricular septal defect, tricuspid valve abnormalities and atrioventricular block.

Molecular mechanism of mechanical stress-induced cardiac hypertrophy.

Mechanical stress is a major cause of cardiac hypertrophy. Although the mechanisms by which mechanical load induces cardiomyocyte hypertrophy have long been a subject of great interest for cardiologists, the lack of a good in vitro system has hampered the understanding of the biochemical mechanisms. For these past several years, however, an in vitro neonatal cardiocyte culture system has made it possible to examine the biochemical basis for the signal transduction of mechanical stress. Passive stretch of cardiac myocytes cultured on silicone membranes activates phosphorylation cascades of many protein kinases including protein kinase C, Raf-1 kinase and extracellular signal regulated kinases, and induces the expression of specific genes as well as an increase in protein synthesis. During that process, the secretion and production of vasoactive peptides such as angiotensin II and endothelin, are increased and they play critical roles in the induction of these hypertrophic responses. Although the involvement of vasoactive peptides in the development of cardiac hypertrophy is clinically important, the "mechanoreceptor" which receives the mechanical stress and converts it into intracellular biochemical signals remained unknown. We have recently obtained evidence suggesting that ion channels and integrins may be the "mechanoreceptor", the activation of which leads to cardiac hypertrophy.

Glomerular overexpression and increased tyrosine phosphorylation of focal adhesion kinase p125FAK in lupus‐prone MRL/MP‐lpr/lpr mice

Much progress has been made in understanding how mammalian cells receive a diverse array of external stimuli and convert them into intracellular biochemical signals. Such efforts have identified a large number of signalling molecules. However, our knowledge is limited as to their pathophysiological role in particular diseases. We demonstrate herein that an integrin-linked signalling molecule, focal adhesion kinase p125FAK (FAK), is overexpressed in glomeruli of lupus-prone MRL/MP-lpr/lpr (MRL-lpr) mouse as compared to its congeneic MRL-+/+ strain. Increased expression was specifically demonstrated in glomeruli but not in other tissues examined. The overexpression was observed in 16-week-old MRL-lpr mice with active nephritis, as well as in younger animals at 4 weeks of age. Thus, the upregulation of FAK clearly preceded the clinical onset of nephritis. FAK in MRL-lpr glomeruli is highly tyrosine phosphorylated and is associated with adapter protein Grb2. Previous in vitro studies have shown that the association of FAK/Grb2 links cell adhesion to the Ras pathway, which ultimately stimulates mitogen-activated protein (MAP) kinase, an important regulator of cell proliferation. In accordance, we observed constitutive MAP kinase activation in MRL-lpr glomeruli. Our findings suggest that signalling pathways involving FAK are activated in MRL-lpr glomeruli, and are likely to play a role in the development and progression of autoimmune-mediated murine nephritis.

Novel cardiovascular actions of the activins.

Proliferation and directed migration of vascular cells are key components in vascular diseases such as atherosclerosis and restenosis following percutaneous transluminal coronary angioplasty. However, the precise cellular and molecular mechanisms involved in the control of vascular cell proliferation or migration at the tissue level remain largely undefined. Molecules contributing to these processes are elaborated by distinct cell types and act in both autocrine and paracrine modes. They include two broad classes, polypeptide growth factors and vasoactive G-protein-coupled receptor (GPCR) agonists. Examples of the former, such as platelet-derived growth factor, bind to and activate cell surface receptor tyrosine kinases, initiating intracellular biochemical signaling pathways associated with cell proliferation or migration. In contrast, recent evidence suggests that vasoactive GPCR agonists (e.g. angiotensin II, endothelin-1, alpha-thrombin) elicit cell growth indirectly by inducing the production of autocrine or paracrine factors in vascular cells. Recent studies have identified activin A as a novel component of conditioned medium obtained from GPCR agonist-stimulated vascular smooth muscle cells (SMCs). Although activin A alone only weakly stimulated rat aortic SMC DNA synthesis, it demonstrated a potent co-mitogenic effect in combination with either epidermal growth factor (EGF) or heparin binding EGF-like growth factor in these cells, increasing DNA synthesis by up to 5- and 4-fold respectively. Furthermore, in a rat carotid-injury model, activin A mRNA was upregulated within 6 h after injury, followed by increases in immunoreactive protein detected in the expanding neointima 7 to 14 days later. Taken together, these results indicate that activin A is a common vascular SMC-derived growth factor induced by vasoactive agonists that may, either alone or in combination with other factors, contribute to fibroproliferative vascular diseases.

T cell receptor (TCR) antagonism without a negative signal: evidence from T cell hybridomas expressing two independent TCRs.

Antagonist peptides inhibit T cell responses by an unknown mechanism. By coexpressing two independent T cell receptors (TCRs) on a single T cell hybridoma, we addressed the question of whether antagonist ligands induce a dominant-negative signal that inhibits the function of a second, independent TCR. The two receptors, Valpha2Vbeta5 and Valpha2Vbeta10, restricted by H-2Kb and specific for the octameric peptides SIINFEKL and SSIEFARL, respectively, were coexpressed on the same cell. Agonist stimulation demonstrated that the two receptors behaved independently with regard to antigen-induced TCR downregulation and intracellular biochemical signaling. The exposure of one TCR (Valpha2Vbeta5) to antagonist peptides could not inhibit a second independent TCR (Valpha2Vbeta10) from responding to its antigen. Thus, our data clearly demonstrate that these antagonist ligands do not generate a dominant-negative signal which affects the responsiveness of the entire cell. In addition, a kinetic analysis showed that even 12 h after engagement with their cognate antigen and 10 h after reaching a steady-state of TCR internalization, T cells were fully inhibited by the addition of antagonist peptides. The window of susceptibility to antagonist ligands correlated exactly with the time required for the responding T cells to commit to interleukin 2 production. The data support a model where antagonist ligands can competitively inhibit antigenic peptides from productively engaging the TCR. This competitive inhibition is effective during the entire commitment period, where sustained TCR engagement is essential for full T cell activation.

The tranquilizing injection of Yersinia proteins: a pathogen's strategy to resist host defense.

Pathogenic bacteria of the genus Yersinia possess a type III secretion apparatus by which they can inject up to six effector proteins into host cells. These so-called effector Yops (Yersinia outer proteins) disrupt cellular immune defense functions such as TNF-alpha release, O2-production or phagocytosis and thereby allow Yersinia to grow extracellularly. Recent findings indicate that the effector Yops are highly active proteins that engage in crucial eukaryotic signaling mechanisms. For instance, the translocated tyrosine phosphatase YopH dephosphorylates the focal adhesion proteins paxillin and p130Cas within target cells. Furthermore, the Yersinia effector YopP is able to induce apoptosis in macrophages presumably by blocking MAP kinase and NFKB mediated signaling events. Here we discuss recent advances concerning the intracellular targets and biochemical signaling mechanisms regulated by the translocated Yersinia effectors.

Current issues in invertebrate phototransduction. Second messengers and ion conductances.

Investigation of phototransduction in invertebrate photoreceptors has revealed many physiological and biochemical features of fundamental biological importance. Nonetheless, no complete picture of phototransduction has yet emerged. In most known cases, invertebrate phototransduction involves polyphosphoinositide and cyclic GMP (cGMP) intracellular biochemical signaling pathways leading to opening of plasma membrane ion channels. Excitation is Ca(2+)-dependent, as are adaptive feedback processes that regulate sensitivity to light. Transduction takes place in specialized subcellular regions, rich in microvilli and closely apposed to submicrovillar membrane systems. Thus, excitation is a highly localized process. This article focuses on the intracellular biochemical signaling pathways and the ion channels involved in invertebrate phototransduction. The coupling of signaling cascades with channel activation is not understood for any invertebrate species. Although photoreceptors have features that are common to most or all known invertebrate species, each species exhibits unique characteristics. Comparative electrophysiological, biochemical, morphological, and molecular biological approaches to studying phototransduction in these species lead to fundamental insights into cellular signaling. Several current controversies and proposed phototransduction models are evaluated.

Ras Activation Is Necessary for Integrin-mediated Activation of Extracellular Signal-regulated Kinase 2 and Cytosolic Phospholipase A2 but Not for Cytoskeletal Organization

Cell adhesion to the extracellular matrix triggers a cascade of intracellular biochemical signals regulated by the integrin family of receptors. Recent evidence suggests that integrin engagement may activate a mitogen-activated protein (MAP) kinase cascade that may cooperate with more clearly defined mitogenic signaling pathways to regulate cell proliferation, adhesion, and migration. Here we report that the adhesion-dependent activation of the MAP kinase Erk2 (extracellular signal-regulated kinase 2) occurs in serum-starved NIH3T3 cells, and that this activation of Erk2 is preceded by the activation of the small GTP-binding protein Ras in fibronectin-adherent cells. Inhibition of Ras signaling by expression of a dominant-inhibitory mutant of Ras (N17Ras) in NIH3T3 cells blocked adhesion-dependent activation of Erk2, although the focal adhesion kinase (FAK) was still activated in these cells. Furthermore, activation of this Ras-MAP kinase pathway activated cytosolic phospholipase A2, leading to the release of arachidonic acid metabolites, and N17Ras also inhibited these events. However, N17Ras expression does not inhibit cell adhesion, spreading, or focal contact and stress fiber formation. These results suggest that, while integrin-dependent activation of this MAP kinase pathway is Ras-dependent, the integrin-dependent activation of FAK and several morphological events are Ras-independent. Thus, integrin-mediated signals involved in regulating cell morphology appear to diverge from those regulating MAP kinase activation at a level upstream of Ras activation.

Positive chronotropic responses of rabbit sino-atrial node cells to flash photolysis of caged isoproterenol and cyclic AMP.

The kinetics of onset and the intracellular biochemical signalling mechanisms which are responsible for the positive chronotropic effect of sympathetic stimulation in rabbit cardiac pacemaker cells were examined by using flash photolysis of caged isoproterenol (ISO) and cyclic AMP (cAMP). When caged ISO (10 microM) was present in the superfusate, a single ultraviolet flash caused gradual increases in the spontaneous beating frequency and action potential height of S-A node cells. Both these effects developed after an initial latency of approximately 5 s. Photorelease of ISO also increased the L-type Ca2+ current (ICa-L) with a time-course similar to that of the changes in action potential waveform and heart rate. All of these ISO-induced effects were blocked completely by 1 microM propranolol, demonstrating that they were beta-adrenergic responses. Flash photolysis of caged cAMP (50 microM) also resulted in increased firing frequency and ICa-L. However, these responses to cAMP developed with little or no latency. Intracellular dialysis with a selective inhibitor of the cAMP-dependent protein kinase, Rp-cAMPS, completely abolished the increase in ICa-L demonstrating that it is mediated exclusively via cAMP-dependent activation of protein kinase A, as opposed to a direct G-protein mediated mechanism.

Mechanotransduction and the functional response of bone to mechanical strain.

Mechanotransduction plays a crucial role in the physiology of many tissues including bone. Mechanical loading can inhibit bone resorption and increase bone formation in vivo. In bone, the process of mechanotransduction can be divided into four distinct steps: (1) mechanocoupling, (2) biochemical coupling, (3) transmission of signal, and (4) effector cell response. In mechanocoupling, mechanical loads in vivo cause deformations in bone that stretch bone cells within and lining the bone matrix and create fluid movement within the canaliculae of bone. Dynamic loading, which is associated with extracellular fluid flow and the creation of streaming potentials within bone, is most effective for stimulating new bone formation in vivo. Bone cells in vitro are stimulated to produce second messengers when exposed to fluid flow or mechanical stretch. In biochemical coupling, the possible mechanisms for the coupling of cell-level mechanical signals into intracellular biochemical signals include force transduction through the integrin-cytoskeleton-nuclear matrix structure, stretch-activated cation channels within the cell membrane, G protein-dependent pathways, and linkage between the cytoskeleton and the phospholipase C or phospholipase A pathways. The tight interaction of each of these pathways would suggest that the entire cell is a mechanosensor and there are many different pathways available for the transduction of a mechanical signal. In the transmission of signal, osteoblasts, osteocytes, and bone lining cells may act as sensors of mechanical signals and may communicate the signal through cell processes connected by gap junctions. These cells also produce paracrine factors that may signal osteoprogenitors to differentiate into osteoblasts and attach to the bone surface. Insulin-like growth factors and prostaglandins are possible candidates for intermediaries in signal transduction. In the effector cell response, the effects of mechanical loading are dependent upon the magnitude, duration, and rate of the applied load. Longer duration, lower amplitude loading has the same effect on bone formation as loads with short duration and high amplitude. Loading must be cyclic to stimulate new bone formation. Aging greatly reduces the osteogenic effects of mechanical loading in vivo. Also, some hormones may interact with local mechanical signals to change the sensitivity of the sensor or effector cells to mechanical load.

Partial T cell signaling: Altered phospho-ζ and lack of zap70 recruitment in APL-induced T cell anergy

Studies of T cell responses to altered peptide ligands (APLs) have provided functional evidence that a T cell receptor (TCR) can interpret subtle changes in its ligand, resulting in different phenotypic outcomes. One dramatic effect of APL stimulation with live antigen-presenting cells (APCs) is the induction of anergy as opposed to proliferation. We investigated the intracellular signaling events involved in generating this unresponsiveness by comparing protein-tyrosine phosphorylation patterns after stimulation with anergy-inducing APL or the immunogenic peptide. In resting T cell clones, presentation with APL/live APC stimulated a unique pattern of TCR phospho-ζ species and a subsequent lack of association with zap70. This demonstrates that the TCR-CD3 complex can engage selective intracellular biochemical signaling pathways as a direct consequence of the nature of the ligand recognized and the initial phosphotyrosine pattern of the TCR-CD3 proteins, leading to different phenotypes.

The phosphoinositide cascade in isolated outer hair cells: possible role as second messenger for motile responses

Extracellular stimuli are encoded at the cell membrane into intracellular biochemical signals that elicit the physiological response of the cell. Recent evidence suggests that pol~hosphoinositides, lipids enriched in the plasma membrane of neural tissues constitute a fundamental transmembrane signalling system for neuromodulators and hormones which elevate intracellular calcium levels. The initial step in their action is thought to be a stimulation of the phosphodiesteratic hydrolysis of membrane-bound phosphatidylinositol bisphosphate liberating diacylglycerol and inositol trisphosphate as second messengers and initiating an intracellular signal cascade. Diacylglycerol activates a specific protein kinase C and inositol t~sphosphate mobilises intracellular calcium triggering the cellular response (Berridge, 1984, Nishizuka, 1984). We have investigated phosphoinositides and this signal cascade in isolated live outer hair cells. The preliminary results indicate that contractility of outer hair cells may be mediated by this second messenger system. Living outer hair cells were isolated by micromanipulation. Viability of the preparation was assured by the ability of the cells to carry out oxidative phosphorylation and synthesize ATP. Furthermore, recent giga-seal whole-cell recordings showed ceil potentials of 70 mV (Zenner et al., 1985). The prerequisite for the existence of the phosphoinositide cascade is the presence of phosphatidylinositol bisphosphate and related lipids in outer hair cells. Radioactive labelling documented a high metabolism of phosphatidylinositol, phosphatidylinositol 4-phosphate (PtdIns 4-f ), phosphatidylinositol 4,5-bisphosphate (PtdIns 4.5-P,) and phosphatidic acid. After 60 min of incubation with [X PJorthophosphate the pol~hosphoinositides constituted 33 f 7% (Ptdins 4.5-P,) and 28 i 7% (Ptdins 4-P) of the total labelled lipids. It has been shown that calcium will trigger a motile response in isolated outer hair cells (Zenner, 1986). A crucial test for a physiological role of phosphoinositides as a second messenger system is to examine whether the putative second messenger inositol trisphosphate (InsP,) will cause a contraction in the absence of added calcium. Cells were permeabilized with 0.1% Triton X-100 (InsPx will not penetrate intact membranes) and InsP, was added. Con~ntrations as low as lop5 M indeed elicited a ion~tudinal motile response observable within 10 s.