Key Signaling Mechanisms Research Articles

The epidermal permeability barrier: from the early days at Harvard to emerging concepts.

The epidermal permeability barrier: from the early days at Harvard to emerging concepts.

Nitric oxide-donating non-steroidal anti-inflammatory drugs: the case of nitroderivatives of aspirin

Nitric oxide (NO) acts as a key signalling mechanism in a number of cells and tissues in the mammalian organism. Modulation of the biosynthesis of NO has emerged to be relevant to the treatment of a variety of human diseases. In the attempt to reduce the serious side effects of non-steroidal anti-inflammatory drugs (NSAIDs), especially in the gastrointestinal tract, a NO-releasing moiety has been linked to conventional NSAIDs. A prototypical example is that of NO-releasing derivatives of aspirin. Thanks to the cytoprotective action of NO such compounds do not produce gastric damage and are emerging as an interesting novel group of drugs for their unique pharmacological properties.

Invited Review: Autocrine/paracrine IGF-I and skeletal muscle adaptation.

This brief review presents the basic premises suggesting that insulin-like growth factor I (IGF-I), functioning in an autocrine/paracrine mode, is an important mediator of skeletal muscle adaptation. Key intracellular signaling mechanisms associated with ligation of the primary IGF-I receptor are highlighted to illustrate the mechanisms by which IGF-I may promote muscle hypertrophy. In addition, a number of recent findings are presented that highlight the potential for interactions between IGF-I-related signaling pathways and intracellular signaling mechanisms activated by cytokines or hormonal systems.

Protein synthesis during LTP: linking synaptic activity to translation

Persistent enhancement of synaptic strength, as in long-term potentiation (LTP), is believed to be important in the regulation of memory storage in the CNS. A hallmark of long-lasting LTP is its requirement for new protein synthesis. Experiments using hippocampal slices have shown that pharmacological inhibitors of translation block long-lasting forms of LTP without affecting early stages of LTP expression. However, such experiments do not reveal how synaptic activity leading to LTP triggers translation in neurons. One possibility is that local dendritic protein synthesis, involving translation in postsynaptic dendrites activated by synaptic stimulation, could provide the proteins necessary for expression of long-lasting forms of LTP. Several proteins that are critical for regulating translational initiation have been identified in mammalian cells. These include mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates the activity of proteins that bind to eukaryotic initiation factor-4E (4E-BPs). Phosphorylation of 4E-BPs by mTOR allows these proteins to dissociate from initiation factor-4E (eIF-4E), thereby initiating translation. Demonstration of the presence of these regulatory proteins at postsynaptic dendritic sites, and their participation in LTP, would represent an important step towards identifying key signalling mechanisms that link LTP-inducing synaptic stimuli to initiation of protein synthesis.

Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes.

Phosphorylation of proteins on serine/threonine residues preceding proline is a key signaling mechanism. The conformation and function of a subset of these phosphorylated proteins is regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser/Thr-Pro bonds. Although young Pin1(-/-) mice have been previously shown to develop normally, we show here that they displayed a range of cell-proliferative abnormalities, including decreased body weight and testicular and retinal atrophies. Furthermore, in Pin1(-/-) adult females, the breast epithelial compartment failed to undergo the massive proliferative changes associated with pregnancy. Interestingly, many of these Pin1-deficient phenotypes such as retinal hypoplasia and mammary gland impairment are also the characteristic of cyclin D1-deficient mice. Cyclin D1 levels were significantly reduced in many tissues in Pin1-deficient mice, including retina and breast epithelial cells from pregnant mice. Moreover, Pin1 directly bound to cyclin D1 phosphorylated on Thr-286-Pro increased cyclin D1 in the nucleus and stabilized cyclin D1. These results indicate that Pin1 positively regulates cyclin D1 function at the transcriptional level, as demonstrated previously, and also through posttranslational stabilization, which together explain why Pin1 loss-of-function phenotypes in the mouse resemble cyclin D1-null phenotypes. Our results provide genetic evidence for an essential role of Pin1 in maintaining cell proliferation and regulating cyclin D1 function.

Pin1-Dependent Prolyl Isomerization Regulates Dephosphorylation of Cdc25C and Tau Proteins

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. Here we show that the major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs.

Atherogenic lipoproteins stimulate mesangial cell p42 mitogen-activated protein kinase.

Previously, it has been shown that atherogenic lipoproteins, through the activation of glomerular cells, stimulate pathobiological processes involved in monocyte infiltration into the mesangium. This study examined the role of LDL and its oxidatively modified variants (mildly oxidatively modified LDL [mm-LDL] and oxidatively modified LDL [ox-LDL]) on the activation of mesangial cell p42 mitogen-activated protein kinase (MAP kinase), a key intracellular signaling mechanism associated with cell proliferation. The incubation of mesangial cells with either LDL, mm-LDL, or ox-LDL induced the activation of MAP kinase dose dependently. The activation of MAP kinase by these lipoproteins in mesangial cells occurred biphasically: initially at 15 min of incubation period and at later time points of 8 to 24 h. No activation of MAP kinase was noted between 30 min (except in LDL) and 6 h. The induction of MAP kinase by both mm-LDL and ox-LDL was greater by 1.5- to 2-fold when compared with LDL. Similarly, these atherogenic lipoproteins stimulated mesangial cell proliferation. Lysophosphatidylcholine, a component of both oxidatively modified variants of LDL, markedly stimulated mesangial cell MAP kinase activity at early incubation times (5 to 30 min) but not at later time points (3 to 24 h), suggesting that lysophosphatidylcholine may, at least in part but not solely, act as an active component of ox-LDL-mediated effects. These data define putative key signal transduction events associated with lipoprotein-mediated induction of mesangial cell proliferation.

Role of intracellular signalling pathways in hydrogen peroxide-induced injury to rat glomerular mesangial cells.

1. Brief exposure of cultured rat glomerular mesangial cells (GMC) to H2O2 in nominally bicarbonate-free solution induced a rapid dose dependent, dantrolene-inhibitable increase in intracellular free Ca2+ from 65 +/- 6 to 203 +/- 14 nmol/L and a prolonged release of [14C]-arachidonic acid [14C]-AA which preceded the onset of cell membrane damage assessed by trypan-blue uptake. 2. Ca2+ responses were potentiated in HCO3-/CO2 containing buffers and reached values of 1145 +/- 100 nmol/L at 1 mmol/L H2O2. In HCO3-/CO2 solutions, but not HEPES buffer, H2O2-induced Ca2+ increases were markedly attenuated by verapamil (100 mumol/L) or removal of extracellular calcium. 3. Enhanced release of [14C]-AA was partially attenuated by inhibitors of key intracellular signalling mechanisms including the phospholipase-A2 (PLA2) inhibitor mepacrine (100 mumol/L), the NADPH oxidase inhibitor diphenyliodonium (10 mumol/L), the mitochondrial calcium-cycling inhibitor ruthenium red (10 mumol/L) and the iron chelator dipyridyl (100 mumol/L). Release was unaffected by protein kinase C inhibition with H7 (100 mumol/L), inositol triphosphate antagonism with neomycin (1 mmol/L) or overnight treatment with the G-protein antagonist pertussis toxin (5 micrograms/mL). 4. Several structurally diverse lipoxygenase inhibitors, including esculetin, baicalein and phenidone, over the dose range 1-100 mumol/L, also prevented [14C]-AA release and markedly protected against cell membrane damage. No drug directly scavenged H2O2 assessed by UV absorption. 5. These results indicate that H2O2 activates in GMC a complex series of interrelated pathological mechanisms which in turn contribute to a prolongation of oxidative damage beyond the time of the initial exposure. These include an increase in intracellular calcium which, depending upon conditions, appears to be mediated by release from intracellular stores as well as Ca2+ entry from the extracellular space. In turn there is a sustained release of arachidonic acid, which may partly depend on prolonged activation of PLA2 but not phospholipase C. 6. Release of [14C]-AA could be attenuated by inhibitors of NADPH oxidase, mitochondrial calcium-cycling, iron chelators and a structurally diverse range of lipoxygenase inhibitors in association with protection from H2O2-mediated cell membrane damage.

Physiological effects of endogenous ouabain: control of intracellular Ca2+ stores and cell responsiveness.

Ouabain is a well-known compound but a newly discovered adrenal cortical hormone that plays a role in cell Na+ regulation and in whole body salt and water balance. Ouabain may also be a paracrine hormone and may be secreted by some central nervous system neurons as well as by other types of cells. This article focuses on the cellular mechanisms that underlie the physiological (and pathophysiological) effects of ouabain. Ouabain directly inhibits the plasmalemmal Na+ pump in a variety of cell types. Low ouabain concentrations cause, in the steady state, a modest rise in the cytosolic Na+ concentration but only a minimal decline in membrane potential. All Na+ gradient-dependent processes may thereby be affected, albeit to only a small extent. Most important, however, is the secondary redistribution of Ca2+, mediated by Na(+)-Ca2+ exchange, that should slightly increase the cytosolic free Ca2+ concentration ([Ca2+]cyt). As a result of Ca2+ sequestration in intracellular stores [the endoplasmic and/or sarcoplasmic reticulum (ER/SR)], however, a new steady state is achieved with a slightly increased [Ca2+]cyt but a substantially augmented Ca2+ store; thus the ER/SR effectively acts as a Ca2+ amplifier. This extra stored Ca2+ is then available for mobilization whenever the cells are activated. Cytosolic Ca2+ is a key signaling mechanism in virtually all cells: it controls numerous physiological processes such as contraction, secretion, and excitability. Thus ouabain may modulate cell responsiveness via its influence on ER/SR Ca2+ stores. With these principles in mind, we examine evidence that endogenous ouabain may play a role in numerous physiological and pathophysiological processes associated with altered fluid and electrolyte metabolism and deviations from the normal blood pressure-blood volume relationship. We discuss the possible participation of ouabain in the regulation of vascular tone and then consider the putative role of ouabain in several forms of hypertension, congestive heart failure, thyroid and adrenocortical dysfunction, and diabetes mellitus, as well as in the adaptation to high altitude.

Modifications of protein-DNA interactions in the proximal promoter of a cell-growth-regulated histone gene during onset and progression of osteoblast differentiation.

A temporal sequence of interrelated cellular, biochemical, and molecular events which occurs during the progressive expression of the differentiated osteoblast phenotype in primary cultures of fetal rat calvarial cells results in the development of a bone-tissue-like organization. This ordered developmental sequence encompasses three periods: proliferation, matrix maturation, and mineralization. Initially, the cells actively proliferate and synthesize type I collagen. This is followed by a period of matrix organization and maturation and then by a period of extracellular matrix mineralization. At the completion of proliferation, when expression of osteoblast phenotype markers such as alkaline phosphatase is observed, the cell-cycle-related histone genes are down-regulated transcriptionally, suggesting that a key signaling mechanism at this transition point involves modifications of protein-DNA interactions in the regulatory elements of these growth-regulated genes. Our results demonstrate that there is a selective loss of interaction of the promoter binding factor HiNF-D with the site II region of an H4 histone gene proximal promoter that regulates the specificity and level of transcription only when the down-regulation of proliferation is accompanied by modifications in the extracellular matrix that contribute to progression of osteoblast differentiation. Thus, this specific loss of protein-DNA interaction serves as a marker for a key transition point in the osteoblast developmental sequence, where the down-regulation of proliferation is functionally coupled to the appearance of osteoblast phenotypic properties associated with the organization and maturation of an extracellular matrix that becomes competent to mineralize.