Karen M. Lounsbury

The Ras superfamily of GTPases.

The Ras superfamily of small GTPases comprises a group of molecular switches that regulate an astonishing diversity of cellular functions. A deep understanding of mitogenesis, cy‐toskeletal organization, vesicle traffic, and nuclear transport now requires the inclusion of the small GTPases as essential components of the molecular machines that drive these processes. The rich complexity of the control mechanisms involved is evidenced by the recent discoveries of GTPase cascades, multiple downstream effectors, and interconnected networks of GTPase‐regulated protein kinase cascades. The 1995 FASEB Summer Conference at Snowmass Village, Colorado, on the Ras GTPase superfamily provided testimony to the broad impact that the study of these proteins continues to exert on cell biology.—Macara, I. G., Lounsbury, K. M., Richards, S. A., McKiernan, C., Bar‐Sagi, D. The Ras superfamily of GTPases. FASEB J. 10, 625‐630 (1996)

Calcium signaling and oxidant stress in the vasculature

Recent evidence suggests that oxidant stress plays a major role in several aspects of vascular biology. Oxygen free radicals are implicated as important factors in signaling mechanisms leading to vascular pathologies such as postischemic reperfusion injury and atherosclerosis. The role of intracellular Ca(2+) in these signaling events is an emerging area of vascular research that is providing insights into the mechanisms mediating these complex physiological processes. This review explores sources of free radicals in the vasculature, as well as effects of free radicals on Ca(2+) signaling in vascular endothelial and smooth muscle cells. In the endothelium, superoxides enhance and peroxides attenuate agonist-stimulated Ca(2+) responses, suggesting differential signaling mechanisms depending on radical species. In smooth muscle cells, both superoxides and peroxides disrupt the sarcoplasmic reticulum Ca(2+)-ATPase, leading to both short- and long-term effects on smooth muscle Ca(2+) handling. Because vascular Ca(2+) signaling is altered by oxidant stress in ischemia-related disease states, understanding these pathways may lead to new strategies for preventing or treating arterial disease.

Coupling of Ca(2+) to CREB activation and gene expression in intact cerebral arteries from mouse : roles of ryanodine receptors and voltage-dependent Ca(2+) channels.

Pathological changes of the vasculature are characterized by changes in Ca(2+) handling and alterations in gene expression. In neurons and other cell types, [Ca(2+)](i) often drives changes in gene expression. However, the relationship between Ca(2+) signaling and gene expression in vascular smooth muscle is not well understood. This study examines the ability of Ca(2+) influx through voltage-dependent, L-type Ca(2+) channels (VDCCs) and Ca(2+) release through ryanodine receptors (RyRs) to activate the transcription factor, cAMP-responsive element binding protein (CREB), and increase c-fos levels in intact cerebral arteries. Membrane depolarization increased the fraction of nuclei staining for phosphorylated CREB (P-CREB) and levels of c-fos mRNA in intact mouse cerebral arteries. Ryanodine, which inhibits RyRs, increased P-CREB staining and c-fos levels. Forskolin, an activator of adenylyl cyclase, and sodium nitroprusside, an NO donor, increased P-CREB and c-fos levels. Nisoldipine, an inhibitor of VDCCs, reversed the effects of depolarization and ryanodine on P-CREB and c-fos levels, but not the effects of forskolin or sodium nitroprusside. Inhibition of Ca(2+)/calmodulin-dependent protein kinase (CaM kinase) blocked increases in P-CREB and c-fos levels seen with membrane depolarization, suggesting that CaM kinase has an important role in the pathway leading from Ca(2+) influx to CREB-mediated changes in c-fos levels. Our data suggest that membrane depolarization increases [Ca(2+)](i) through activation of VDCCs, leading to increased P-CREB and c-fos, and that RyRs have a profound effect on this pathway by indirectly regulating Ca(2+) entry through VDCCs. These results provide the first evidence of Ca(2+) regulation of CREB and c-fos in arterial smooth muscle.

Gender differences in coronary artery diameter reflect changes in both endothelial Ca2+ and ecNOS activity.

Elevation of nitric oxide (NO) release from the vascular endothelium may contribute to some of the gender-associated differences in coronary artery function. The mechanisms by which gender affects NO release from the endothelium of coronary arteries are not known. In this study, endothelial function was examined in pressurized coronary arteries from female and male rats. Diameter and endothelial cell intracellular Ca2+ concentration ([Ca2+]i) in intact arteries, as well as enzymatic activity of endothelial constitutive nitric oxide synthase (ecNOS) in arterial lysates, was measured. Elevation of intravascular pressure to 60 mmHg constricted coronary arteries from female animals less than coronary arteries from male animals (18% and 31% constriction, respectively). The increased arterial diameter of coronary arteries from females was associated with elevated endothelial [Ca2+]i (female 174 nM, male 90 nM; P < 0.001). Elevation of Ca2+ activated ecNOS with a similar slope and half-activation constant ( approximately 160 nM) for both female and male coronary arteries. However, at [Ca2+] > 100 nM, ecNOS activity was significantly higher in coronary arteries from female rats compared with their male equivalents (P < 0.01). Maximal activity for ecNOS at saturating Ca2+ (300 nM) was 37% higher in coronary arteries from female animals compared with male animals (P < 0.05). Thus elevated [Ca2+]i in the endothelium of female coronary arteries alone is predicted to increase the production of NO (by nearly 2-fold). This gender difference combined with increased ecNOS activity at a given [Ca2+] in females indicates that tonic NO production should be nearly threefold greater in female coronary arteries compared with male coronary arteries. We conclude that, in the regulation of endothelial Ca2+ and ecNOS, gender differences contribute significantly to the overall decrease in myogenic tone observed in coronary arteries of females.

Hypoxia‐mediated biological control

When oxygen demand is greater than oxygen supply, cells need to rapidly adjust their metabolism in order for the tissue to survive. Oxygen sensing by an organism influences a host of processes including growth, development, metabolism, pH homeostasis, and angiogenesis. Hypoxia also contributes to a wide number of human diseases including vascular disease, inflammatory conditions and cancer. Recently, major advances have been made in understanding the response of cells and tissues to hypoxia with the goal of providing mechanistic insight and novel therapeutic targets. In this article we review both the normal biological effects of hypoxia as well as the alterations that occur in specific disease conditions with an emphasis on the cell signaling and gene transcription mechanisms that underlie the changes associated with chronic hypoxia. Comparisons of studies in the fields of cardiac ischemia and tumor angiogenesis reveal the complexities within the microenvironment that control responses to hypoxia. It is clear that more interaction between researchers in these fields will improve the development of therapies that either promote or prevent hypoxic responses. J. Cell. Biochem. 112: 735–744, 2011.

Mutations within the Ran/TC4 GTPase. Effects on regulatory factor interactions and subcellular localization.

Ran, a member of the Ras superfamily of GTPases, is predominantly localized in the nucleus and is a necessary component in the active transport of proteins through nuclear pores. Disruption of Ran function affects the regulation of mitosis, DNA synthesis, and RNA processing and export. To explore the mechanisms of Ran function, mutants of the Ran GTPase were characterized, several of which are capable of dominantly interfering with nuclear protein import. Unlike wild-type Ran, the putative gain-of-function mutant (G19V Ran) was not sensitive to the exchange factor, RCC1. In addition the G19V Ran and effector domain mutants (L43E and E46G Ran) were not sensitive to the GTPase-activating protein, Fug1. Epitope-tagged G19V Ran and L43E Ran isolated from transfected BHK21 cells were each about 50% GTP-bound, whereas the wild-type and a C-terminal deletion mutant (Δ-DE Ran) were primarily bound to GDP. While G19V Ran interacted with known Ran-binding proteins and with an isolated Ran-binding domain, the T24N Ran did not, and binding by L43E Ran was substantially reduced. Wild-type HA1-tagged Ran expressed in BHK21 cells was nuclear, whereas the G19V, T24N, L43E, and E46G forms of Ran were predominantly localized at the nuclear envelope, and Δ-DE Ran was primarily cytosolic. Similar results were observed when permeabilized BHK21 cells were incubated with extracts of COS cells expressing the mutants. Thus mutations that affect the interaction of Ran with regulatory proteins and effectors can disrupt the normal subcellular localization of Ran, lending support for the current model of Ran-mediated nuclear import.

Nitric oxide and reactive oxygen species exert opposing effects on the stability of hypoxia-inducible factor-1α (HIF-1α) in explants of human pial arteries

Hypoxia induces angiogenesis, partly through stabilization of hypoxia‐inducible factor‐1α (HIF‐ 1α), leading to transcription of pro‐angiogenic factors. Here we examined the regulation of HIF‐ 1α by hypoxia and nitric oxide (NO) in explants of human cerebrovascular smooth muscle cells. Cells were treated with NO donors under normoxic or hypoxic (2% O2) conditions, followed by analysis of HIF‐1α protein levels. Treatment with the NO donor sodium nitroprusside reduced levels of HIF‐1α, whereas NO donors, NOC‐18 and S‐nitrosoglutathione, increased HIF‐1α levels. SIN‐1, which releases both NO and superoxide (O2•¯), reduced HIF‐1α levels, suggesting that inhibitory NO donors may elicit effects through peroxynitrite (ONOO•¯). O2•¯ generation by xanthine/xanthine oxidase also reduced HIF‐1α levels, confirming an inhibitory role for reactive oxygen species (ROS). Furthermore, superoxide dismutase increased HIF‐1α levels, and the NO scavenger carboxy‐PTIO reversed HIF‐1α stabilization by NO donors. Effects on HIF‐1α levels correlated with vascular endothelial growth factor transcription but did not affect HIF‐1α transcription, as measured by RT‐PCR and luciferase‐reporter assays. The results indicate that HIF‐1α is stabilized by agents that produce NO and reduce ROS but destabilized by agents that increase ROS, including O2•¯ and ONOO•¯. Thus we propose that the effect of NO on HIF‐1α signaling is critically dependent on the form of NO and the physiological environment of the responding cell.

Excitation–transcription coupling in smooth muscle

Calcium (Ca2+) signals affect virtually every biological process, including both contraction and gene transcription in smooth muscle. Ca2+‐regulated gene transcription is known to be important for both physiological and pathological responses in smooth muscle. The aim of this review is to discuss the current understanding of gene transcription regulated by excitation through Ca2+ signalling using a comparison of the two most characterized Ca2+‐regulated transcription factors in smooth muscle, Ca2+–cyclic AMP response element binding protein (CREB) and nuclear factor of activated T‐cells (NFAT). Recent studies have shown commonalities and differences in the regulation of CREB and NFAT through both voltage‐ and non‐voltage‐gated Ca2+ channels that lead to expression of smooth muscle cell specific differentiation markers as well as markers of proliferation. New insights into the regulation of specific genes through companion elements on the promoters of Ca2+‐regulated genes have led to new models for transcriptional regulation by Ca2+ that are defined both by the source and duration of the Ca2+ signal and the composition of enhancer elements found within the regulatory regions of specific genes. Thus the combination of signalling pathways elicited by particular Ca2+ signals affect selective promoter elements that are key to the ultimate pattern of gene transcription.

Store-Operated Ca2+ Entry Activates the CREB Transcription Factor in Vascular Smooth Muscle

Ca2+-regulated gene transcription is a critical component of arterial responses to injury, hypertension, and tumor-stimulated angiogenesis. The Ca2+/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, is activated by Ca2+-induced phosphorylation. Multiple Ca2+ entry pathways may contribute to CREB activation in vascular smooth muscle including voltage-dependent Ca2+ channels and store-operated Ca2+ entry (SOCE). To investigate a role for SOCE in CREB activation, we measured CREB phosphorylation using immunofluorescence, intracellular Ca2+ levels using a fluorescence resonance energy transfer (FRET)–based Cameleon indicator, and c-fos transcription using RT-PCR. In this study, we report that SOCE activates CREB in both cultured smooth muscle cells and intact arteries. Depletion of intracellular Ca2+ stores with thapsigargin increased nuclear phospho-CREB levels, intracellular Ca2+ concentration, and transcription of c-fos. These effects were abolished by inhibiting SOCE through lowering extracellular Ca2+ concentration or by application of 2-aminoethoxydiphenylborate and Ni2+. Inhibition of Ca2+ influx through voltage-dependent Ca2+ channels using nimodipine partially blocked intact artery responses, but was without effect in cultured smooth muscle cells. Our findings indicate that Ca2+ entry through store-operated Ca2+ channels leads to CREB activation, suggesting that SOCE contributes to the regulation of gene expression in vascular smooth muscle.

Ran binding domains promote the interaction of Ran with p97/beta-karyopherin, linking the docking and translocation steps of nuclear import.

Nuclear protein import is accomplished by two sequential events: docking at the nuclear pore complex followed by ATP-dependent translocation across the nuclear envelope. Docking of nuclear targeted proteins requires a 56-kDa nuclear localization signal receptor (α-karyopherin, importin-α, SRP1α) and a 97-kDa protein (β-karyopherin, importin-β). Components necessary for translocation include the Ran/TC4 GTPase and NTF2/B-2. The functions of these factors at a molecular level remain unclear. We have now found that a complex of Ran, in the GTP-bound state, with either the Ran binding protein, RanBP1, or an isolated Ran binding domain binds with high affinity and specificity to β-karyopherin to form a ternary complex. We find that a C-terminal truncation mutant of Ran, Δ-DE Ran, also binds to β-karyopherin and that Δ-DE Ran can associate with a cytosolic, multiprotein complex that contains β-karyopherin and another Δ-DE Ran binding protein of 115/120 kDa. These data suggest a physical link between docking and translocation mediated by a Ran GTPase-Ran binding protein complex.