|T H E N I H C A T A L Y S T||S E P T E M B E R - O C T O B E R 1 9 9 8|
|P E O P L E|
Toren Finkel received his M.D. and Ph.D. from Harvard Medical School in in Boston in 1986. He completed a residency in internal medicine at the Massachusetts General Hospital in Boston and a cardiology fellowship at Johns Hopkins University in Baltimore before joining the NHLBI Cardiology Branch in 1992. He is currently a senior investigator and chief of the Cellular and Molecular Biology Section in that branch.
Atherosclerotic heart disease remains the leading cause of morbidity and mortality in the Western world. Over the past 20 years, many studies have demonstrated the role of blood pressure, smoking, cholesterol, and other factors in disease progression, but relatively little is known about how these systemic factors result in localized plaque formation.
Before the development of atherosclerotic plaque in both animal models and human subjects, however, there appears to be an increase in the production of reactive oxygen species (ROS) from the vessel wall. My lab has focused on how these ROS are generated and regulated in nonphagocytic cells and what intracellular signaling pathways they seem to regulate.
Early in our studies, we observed that vascular smooth muscle cells stimulated by platelet-derived growth factor (PDGF) produce a large and rapid but transient increase in intracellular H2O2. We were able to demonstrate that this increase in ROS was essential for PDGF signal transduction and, in particular, for growth factorstimulated tyrosine phosphorylation. Results from our lab and others have since suggested that the burst of ROS is not confined to smooth muscle cells or PDGF, but occurs with a variety of ligands in a multitude of cells.
We next were able to demonstrate the role of the small GTPases ras and rac1 in the regulation of ligand-stimulated ROS. This was particularly interesting because rac proteins were already known to regulate ROS production in nonphagocytic cells. Nonetheless, these results suggested that the ras superfamily of proteins might function to regulate the balance of oxidation and reduction, that is, the redox state, of the cell.
Not only does our work suggest that ROS play a role in growth-factor signaling, we have also demonstrated that ROS serve as mediators of apoptosis. Using an adenovirus to deliver wild-type p53, we have demonstrated that p53 expression results in an increase in ROS, which is needed to initiate apoptosis. More recently, we have shown that ROS also mediate certain aspects of senescence.
Taken together, these results suggest that the redox state of the cell is actively regulated and plays an important role in a variety of pathways as diverse as growth, death, and senescence. The mechanism by which small diffusible molecules like H2O2 can regulate targeted pathways is not yet fully understood. We are currently seeking to identify specific intracellular protein targets of ROS. Furthermore, we hope to relate these findings back to the vessel wall to understand how continuously elevated concentrations of ROS contribute to atherosclerotic disease progression.
My team investigates the influence of the lipid matrix on the function of neural receptor proteins, in particular, the influence of high concentrations of polyunsaturated fatty acids, such as the unsaturated docosa-hexaenoic acid (DHA, 22:6n3) with six double bonds. The phospholipids of brain synaptosomes and the retina contain 30 to 50 mol% DHA as fatty acids. Several lines of evidence suggest that high DHA concentrations are necessary to achieve full activity of certain neural membrane receptors.
I have applied recent developments in NMR spectroscopy to the study of membrane structure and dynamics to obtain a better description of membrane properties that modulate membrane receptor function. Modern NMR techniques require only milligram-size samples of membrane material and are compatible with investigation of complex biological membranes containing mixtures of lipids and proteins at physiological conditions. With atomic resolution, we are able in many instances to pinpoint the location of membrane molecules in the lipid matrix.
For example, we determined that short-chain alcohols such as ethanol locate preferentially near the membrane-water interface and lower interfacial energy of lipids and proteins. These techniques enable a more detailed description of membrane biophysical properties, including parameters that describe the energy of elastic membrane deformation, that can be linked to the membrane receptors' structural transitions during excitation.
The alteration of membrane mechanical properties is one possible role of lipid polyunsaturation. There has been controversy concerning the nature of the perturbation of membrane elasticity induced by DHA chains. The six methylene-interrupted cis double bonds within DHA's 22-carbon unit reduce the number of degrees of freedom for structural transitions, a finding that has led some investigators to suggest that these chains have a specific, rigid conformation. However, this hypothesis is at variance with experimental results. We determined, for the first time, a large number of parameters that describe orientation and motion of individual DHA chain segments in biomembranes, and we measured by X-ray diffraction average DHA chain length and the molecular cross-sectional area. The data indicated an unexpected high deformability of DHA chains. This information provided constraints by which to examine DHA conformations proposed by molecular modeling studies to determine their correlation with experimental data.
Our results suggest that DHA chains in membranes prefer looped conformations and undergo rapid structural transitions, providing increased flexibility to receptor-rich neural membranes. Moreover, our research is beginning to uncover a framework within which the biophysical properties and functions of membrane lipids can be understood in terms of their degree of unsaturation, a discrimination that nature clearly makes.
I have a long-standing interest in how cellular diversity is established and maintained. As a confirmed Drosophila geneticist, I have used the sophisticated genetics of the fruit fly Drosophila melanogaster to identify and characterize the genes involved in one particular developmental step, the specification of segmental identity in the fly. Segmental identity is specified by the homeotic genes, the Drosophila homologues of the HOX genes of vertebrates. Because the homeotic genes have 100-kb cis-regulatory regions that control their developmental expression patterns, a large number of proteins are required to specify and maintain expression. I have concentrated on identifying and characterizing two groups of genes that function to maintain patterns of gene activity, either repression or activation. These two groups of genes, the Polycomb and trithorax, are conserved between Drosophila and humans.
My colleagues and I have identified more than a dozen new Polycomb and trithorax group genes using genetic screens. We have cloned and characterized several of these new genes, including the brahma (brm) gene. We showed that the BRM protein functions as the ATPase subunit of a 2-megadalton protein complex. This complex is conserved from yeast to humans and appears to be a chromatin remodeling machine. We have shown that BRM is required to maintain expression not only of the homeotic genes but of several other important developmental genes as well. It is not required for expression of all Drosophila genes, however.
We are currently trying to understand what recruits this large protein complex to its target genes. We have identified putative brm-response elements in the cis-regulatory regions of two of the target homeotic genes and have used genetic screens to identify other proteins that interact with BRM in regulating these target genes. BRM appears to interact with different sets of proteins at these two cis-regulatory elements.
Complementing our work on the proteins that maintain transcriptional activation of the homeotic genes, we are now also identifying and characterizing proteins required to maintain repression of these genes. One of these new repressors appears to be part of a histone deacetylase complex, which suggests a role for histone deacetylation in maintaining repression of the homeotic genes.
We began exploring fruit fly development in the hope that our observations would elucidate how genes control human development. Because not only the homeotic genes but also the proteins that regulate their function appear to be conserved between flies and humans, we are finding that our hopes have not been misplaced. Moreover, the emergence of increasingly sophisticated molecular genetic approaches in Drosophila bolsters our confidence that this research will continue to expand our understanding of human developmental processes, both normal and defective.
My current research uses RNA polymerase (pol) III and its associated factors as a model to explore the mechanisms that control transcription in eukaryotes and to elucidate how the expression of certain small RNA genesnamely tRNA and Alu family retroposonsare regulated. Pol III also synthesizes a variety of other transcripts, including 5S rRNA and U6 small nuclear (sn) RNA, but their promoter genes are organized differently from those of tRNA and Alu and engage different arrays of pol III transcription factors. Several viruses rely on pol III for the expression of their small RNA genes, most notably adenovirus, whose virus-associated [VA] RNA products modulate the host-cell translational machinery. A growing number of pathogenic viruses not only use pol III for expression of their genes, but also encode proteins that modulate certain host-cell pol IIIassociated factors.
Nearly one million Alu sequences constitute a complex family of mobile elements that "retro"-transpose through their small RNA transcripts, sometimes causing recognizable genetic disorders in humans. These sequences, like the family of tRNA genes, contain pol III promoters within their transcribed region, a feature that endows newly inserted copies with the potential for transcriptional competence. However, unlike other pol IIItranscribed sequences, Alu elements are maintained in a transcriptionally inactive state, becoming activated by viral infection, heat shock, and translational stress. The mechanisms that regulate Alu transcription and the consequences of their expression are largely undefined.
Assembly of a transcription complex on the promoter of a target gene is a key determinant of eukaryotic gene transcriptionbut it is not the only one. My laboratory has shown that control can also occur at the levels of transcription termination and reinitiation. Efficient reinitiation is especially important for tRNA and 5S rRNA genes that must be transcribed at high levels. We have focused on the human La protein, an abundant nuclear phosphoprotein that is recognized as a self-antigen in patients suffering from autoimmune disorders such as systemic lupus erythematosus and Sjögren's syndrome. We have shown that La facilitates transcriptional termination and reinitiation by pol III. Moreover, as an RNA-binding protein that remains associated with nascent pol III transcripts after their synthesis, La also controls the post-transcriptional processing of these RNAs. We have shown that human La is phosphorylated on serine 366, an evolutionarily conserved casein kinase II phosphorylation site, and that this process can regulate La's ability to modulate transcription as well as RNA processing. Thus, the human La phospho-protein can coordinate and regulate transcriptional and post-transcriptional steps in RNA biogenesis.
Our team has developed a pol III transcription-termination reporter gene in the fission yeast Schizosaccharomyces pombe for use in our continuing studies. We use a tRNA opal suppressor capable of suppressing a nonsense codon so that the mRNA can encode a colorimetric metabolic marker (Ade6-704). We plan to examine intracellular signals that can integrate the phosphorylation status of La with pathways relating to other aspects of cell biology and proliferation. Our goal is to use information gained from this system to advance our understanding of gene regulation and cell growth in humans.
My interests lie in the area of cognitive neuroscience, specifically as it relates to understanding perceptual and memory systems. My recent research at NIMH uses functional brain imaging technologies, positron emission tomography, and function magnetic resonance imaging to evaluate the functional neuroanatomy of semantic memorya specific type of memory system that includes the information stored in our brain about the meaning of words and objects.
Our earlier studies sought to clarify word comprehension and word-finding problemssuch as an inability to retrieve object namesin patients with Alzheimer's disease. We learned that such difficulties are related to a loss of information about the features and attributes that define an object and differentiate it from other objects within the same semantic category (for example, information about the features that distinguish a tiger from a leopard or a pair of pliers from a wrench).
Using functional brain imaging with normal subjects, we have been able to demonstrate that information about different types of features, such as an object's typical shape and color, is not stored as a whole unit in a specific place in the brain. Rather, this information is distributed in the brain and organized into a network of discrete cortical regions: Different features and attributes are stored near the regions of the brain that mediate perception of those attributes. Thus, for example, knowledge of object color is stored in a region of the brain adjacent to the areas that mediate perception of color, whereas knowledge about object motion is stored adjacent to areas that mediate motion perception. These findings demonstrate a close link between areas of the brain that mediate perception of different visual features (form, color, and motion) and the regions of the brain where we store these types of information. Additional studies have shown that a similar link exists between brain regions that subserve motor performance and stored knowledge about how objects are used. Thus, the organization of semantic information parallels the organization of the sensory and motor systems in the primate brain.
These studies have provided us with a deeper understanding of object-recognition, object-naming, and language-comprehension problems in a variety of brain disorders, including Alz-heimer's disease and related dementias. They have also provided us with a framework for understanding the etiology of category-specific knowledge disorders that result from focal brain lesions (for example, how one patient can have problems naming and retrieving information about a single category of objects, such as four-legged animals, and another patient can have a deficit limited to naming and knowing about tools). This framework is based on the premise that the distinction between members of different categories of objects depends on access to information about different types of features. More generally, these studies have provided us with a means for asking questions about the broader issue of how information is stored and organized in the human brain.
The research in my laboratory has focused on understanding the mechanisms and functions of calcium signaling in hypothalamic and pituitary cells.
Earlier investigations revealed that two calcium-signaling pathways operate in hypothalamic and pituitary cells: plasma membrane (PM)- and endoplasmic reticulum (ER)- derived. I have shown that these cells express a set of voltage-gated channels that drive spontaneous action potentials, leading to cytosolic calcium fluctuations (the PM oscillator). The major thrust of these investigations was on the role of dihydropyridine-sensitive channels in calcium signaling and the mechanism of their activation and inactivation.
ER-derived calcium signaling in hypothalamic and pituitary cells is activated by several hypothalamic calcium-mobilizing agonists. Agonist-induced calcium release from the ER is mediated by inositol 1,4,5-trisphosphate and leads to frequency-modulated oscillatory calcium signaling in gonadotrophs (the ER oscillator) and to nonoscillatory amplitude-modulated calcium signaling in lacto-trophs (lactotrophs and gonadotrophs are pituitary cells that produce hormones that control lactation and ovulation and spermatogenesis, respectively).
I have characterized the role of several intracellular elements involved in the regulation of the oscillatory vs. nonoscillatory calcium response, such as inositol 1,4,5-trisphosphate receptor channels and the ER calcium pump. I have also studied the coupling of PM and ER oscillators during agonist stimulation. These experimental studies led to the development of quantitative mathematical modelsone for the PM oscillator and one for the ER oscillatoras well as a coupled model that describes the effect of the PM oscillator on depletion and repletion of the ER calcium pool. Functional studies have shown that both hormone release and gene transcription can be activated by calcium-mobilizing agonists when the PM oscillator is rendered inoperative by the depletion of extracellular calcium. In the absence of an agonist, spontaneous activity of the PM oscillator is sufficient to trigger hormone release and early-response gene expression in lactotrophs but not in gonadotrophs.
Current investigations focus on two families of plasma-membrane calcium channels. We recently found that ATP- gated purinergic-receptor (P2X) channels are expressed in pituitary cells and have the capacity to modulate PM- and ER-derived calcium signals and secretion. The primary P2X gene transcripts in pituitary cells undergo extensive al ternative splicing, generating several isoforms. We have identified the amino-acid residues contributing to the desensitization of the P2X2 subtype of these channels, or the protective attenuation of response during prolonged ATP stimulation. The finding that these residues are also expressed in all slowly (but not rapidly) desensitizing P2X channels suggests that a common mechanism controls the rate of cationic influx through these channels, a hypothesis that will be tested in the near future.
We recently identified a novel calcium-influx pathway in several excitable cells that is activated by depletion of the ER calcium pool in a manner comparable to that observed in nonexcitable cells. In neuroendocrine cells, these calcium-influx channels also depolarize the plasma membrane to generate action potentials or to increase the frequency of spiking in spontaneously active cells. Our current investigations are directed toward characterizing the electrophysiological properties of these channels in hypothalamic and pituitary cells, as well as the mechanism of synchronization of calcium release from intracellular stores and action-potentialdriven calcium influx.
Our recent finding that endothelin, unlike other calcium-mobilizing agonists, induces a prolonged inhibition of electrical activitywith associated decreases in calcium influx and cytosolic calcium, depletion of the ER calcium pool, and inhibition of prolactin releasepiqued our interest. Preliminary pharmacological investigations suggest that a novel endothelin receptor is expressed in lactotrophs. We are now attempting to clone this receptor. Functional characterization and coupling of this novel receptor to intracellular messengers will follow.
Another of our objectives is to understand the link between the cellular functions and dynamics of calcium signaling in isolated and interconnected cells. The studies will focus on physiological requirements for the specific pattern of calcium signaling (oscillatory vs. nonoscillatory), the source of calcium (PM- and ER-derived), the threshold calcium concentration needed to activate a specific cellular process, and the role of calcium signals in synchronization of cellular activity among neural and nonneural networks.