Jerry B. Lingrel

Kruppel-like factor 2 regulates thymocyte and T-cell migration

Mammalian Kruppel-like transcription factors are implicated in regulating terminal differentiation of several tissue types. Deficiency in Kruppel-like factor (KLF) 2 (also known as LKLF) leads to a massive loss of the peripheral T-cell pool, suggesting KLF2 regulates T-cell quiescence and survival. Here we show, however, that KLF2 is essential for T-cell trafficking. KLF2-deficient (Klf2-/-) thymocytes show impaired expression of several receptors required for thymocyte emigration and peripheral trafficking, including the sphingosine-1-phosphate (S1P) receptor S1P1, CD62L and β7 integrin. Furthermore, KLF2 both binds and transactivates the promoter for S1P1—a receptor that is critical for thymocyte egress and recirculation through peripheral lymphoid organs. Our findings suggest that KLF2 serves to license mature T cells for trafficking from the thymus and recirculation through secondary lymphoid tissues.

Identification of a Specific Role for the Na,K-ATPase α2 Isoform as a Regulator of Calcium in the Heart

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

Molecular genetics of Na,K-ATPase.

Researchers in the past few years have successfully used molecular-genetic approaches to determine the primary structures of several P-type ATPases. The amino-acid sequences of distinct members of this class of ion-transport ATPases (Na,K-, H,K-, and Ca-ATPases) have been deduced by cDNA cloning and sequencing. The Na,K-ATPase belongs to a multiple gene family, the principal diversity apparently resulting from distinct catalytic alpha isoforms. Computer analyses of the hydrophobicity and potential secondary structure of the alpha subunits and primary sequence comparisons with homologs from various species as well as other P-type ATPases have identified common structural features. This has provided the molecular foundation for the design of models and hypotheses aimed at understanding the relationship between structure and function. Development of a hypothetical transmembrane organization for the alpha subunit and application of site-specific mutagenesis techniques have allowed significant progress to be made toward identifying amino acids involved in cardiac glycoside resistance and possibly binding. However, the complex structural and functional features of this protein indicate that extensive research is necessary before a clear understanding of the molecular basis of active cation transport is achieved. This is complicated further by the paucity of information regarding the structural and functional contributions of the beta subunit. Until such information is obtained, the proposed model and functional hypotheses should be considered judiciously. Considerable progress also has been made in characterizing the regulatory complexity involved in expression of multiple alpha-isoform and beta-subunit genes in various tissues and cells during development and in response to hormones and cations. The regulatory mechanisms appear to function at several molecular levels, involving transcriptional, posttranscriptional, translational, and posttranslational processes in a tissue- or cell-specific manner. However, much research is needed to precisely define the contributions of each of these mechanisms. Recent isolation of the genes for these subunits provides the framework for future advances in this area. Continued application of biochemical, biophysical, and molecular genetic techniques is required to provide a detailed understanding of the mechanisms involved in cation transport of this biologically and pharmacologically important enzyme.

Erythroid-specific expression of human beta-globin genes in transgenic mice.

Transgenic mice carrying human beta‐globin genes were produced by microinjecting linear DNA molecules containing cloned beta‐globin genes with up to 4300 bp of 5′‐flanking sequence and 1700 bp of 3′‐flanking sequence. Most (15 of 20) of these transgenic mice expressed the human beta‐globin genes in blood cells and the level of expression in some mice was comparable with that obtained from endogenous beta‐globin genes. Human beta‐globin gene expression appeared to be restricted to cells of the erythroid lineage and was first detected between 11 and 14 days of development, in parallel with mouse beta‐globin. Constructs with as little as 48 bp of 5′‐flanking sequence also appeared to be expressed appropriately. The mRNA transcripts had correct 5′ ends and directed human beta‐globin synthesis in reticulocyte lysates. Human beta‐globin protein was detectable in mature erythrocytes from progeny of one of these mice. The frequency and extent of expression was severely depressed when the procaryotic vector DNA was not removed prior to microinjection.

Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family.

We have identified and characterized the gene for a novel zinc finger transcription factor which we have termed lung Krüppel-like factor (LKLF). LKLF was isolated through the use of the zinc finger domain of erythroid Krüppel-like factor (ELKF) as a hybridization probe and is closely related to this erythroid cell-specific gene. LKLF is expressed in a limited number of tissues, with the predominant expression seen in the lungs and spleen. The gene is developmentally controlled, with expression noted in the 7-day embryo followed by a down-regulation at 11 days and subsequent reactivation. A high degree of similarity is noted in the zinc finger regions of LKLF and EKLF. Beyond this domain, the sequences diverge significantly, although the putative transactivation domains for both LKLF and EKLF are proline-rich regions. In the DNA-binding domain, the three zinc finger motifs are so closely conserved that the predicted DNA contact sites are identical, suggesting that both proteins may bind to the same core sequence. This was further suggested by transactivation assays in which mouse fibroblasts were transiently transfected with a human beta-globin reporter gene in the absence and presence of an LKLF cDNA construct. Expression of the LKLF gene activates this human beta-globin promoter containing the CACCC sequence previously shown to be a binding site for EKLF. Mutation of this potential binding site results in a significant reduction in the reporter gene expression. LKLF and EKLF can thus be grouped as members of a unique family of transcription factors which have discrete patterns of expression in different tissues and which appear to recognize the same DNA-binding site.

Deficiency in Na,K-ATPase α Isoform Genes Alters Spatial Learning, Motor Activity, and Anxiety in Mice

Several disorders have been associated with mutations in Na,K-ATPase α isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimers disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the α1 isoform is found in many cell types, the α2 isoform is predominantly expressed in astrocytes, and the α3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the α2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the α3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous α1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the α2 and α3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders.

Na, K-ATPase: Isoform Structure, Function, and Expression

An interesting feature of the Na,K-ATPase is the multiplicity of α and β isoforms. Three isoforms exist for the α subunit, α1, α2, and α3, as well for the β subunit, β1, β2, and β3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the α subunit are present in the brain, while only α1 is present in kidney and lung, and α2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na+ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the α subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.

The synthesis of mouse hemoglobin chains in a rabbit reticulocyte cell-free system programmed with mouse reticulocyte 9S RNA

Abstract The 9S RNA fraction of mouse reticulocyte polysomes has been purified and shown to direct the synthesis of mouse hemoglobin β- chains. This has been accomplished by adding mouse 9S RNA to a rabbit reticulocyte cell-free system and incubating in the presence of labeled leucine. After the incubation, carrier mouse hemoglobin was added, globin prepared and mouse β-chains isolated by column chromatography. Labeling of the mouse β-chains in the rabbit cell-free system was strictly dependent upon addition of mouse 9S RNA, demonstrating that the fraction contains the mRNA for mouse hemoglobin β-chains. This is the first definitive demonstration of protein synthesis in a mammalian cell-free system under the direction of a mRNA isolated from a different mammalian species.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long‐term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain‐like compound (EOLC) and induces salt‐dependent hypertension mediated by Na+/Ca2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na+ pump α1 (α1+/−) or α2 (α2+/−) catalytic subunits. Both low‐dose ouabain (1–100 nm; inhibits only α2) and high‐dose ouabain (≥1 μm; inhibits α1) elevate myocyte Ca2+ and constrict arteries from α1+/−, as well as α2+/− and wild‐type mice. Nevertheless, only mice with reduced α2 Na+ pump activity (α2+/−), and not α1 (α1+/−), have elevated blood pressure. Also, isolated, pressurized arteries from α2+/−, but not α1+/−, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB‐R7943) normalize myogenic tone in ouabain‐treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2+/− arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt‐dependent and ouabain‐induced hypertension. Thus, chronically reduced α2 activity (α2+/− or chronic ouabain) apparently regulates myogenic tone and long‐term blood pressure whereas reduced α1 activity (α1+/−) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt‐dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na+ pump activity and promoting Ca2+ entry via NCX in myocytes.

Tissue distribution of mRNAs encoding the α isoforms and β subunit of rat Na+,K+-ATPase

The tissue distribution of the multiple forms of rat Na+,K+-ATPase was examined at the molecular level with cDNA probes specific for the alpha, alpha (+), alpha III and beta subunit mRNAs. Northern and slot blot analyses demonstrate that these mRNAs are produced in a tissue-specific manner. RNAs encoding the alpha (+) isoform are detected in kidney, brain, heart, adipose, muscle, stomach and lung, whereas alpha III RNA is detected in brain, stomach and lung. Both alpha and beta mRNAs are present in all the tissues studied, although at very different levels. Examination of heart tissue in greater detail demonstrates that the levels of mRNA encoding the alpha subunit are greater in the atria than in the ventricles, while the converse is true for alpha (+).