The cardiac glycoside binding site on the Na,K-ATPase alpha2 isoform plays a role in the dynamic regulation of active transport in skeletal muscle.

The physiological significance of the cardiac glycoside-binding site on the Na,K-ATPase remains incompletely understood. This study used a gene-targeted mouse (alpha2(R/R)) which expresses a ouabain-insensitive alpha2 isoform of the Na,K-ATPase to investigate whether the cardiac glycoside-binding site plays any physiological role in active Na(+)/K(+) transport in skeletal muscles or in exercise performance. Skeletal muscles express the Na,K-ATPase alpha2 isoform at high abundance and regulate its transport over a wide dynamic range under control of muscle activity. Na,K-ATPase active transport in the isolated extensor digitorum longus (EDL) muscle of alpha2(R/R) mice was lower at rest and significantly enhanced after muscle contraction, compared with WT. During the first 60 s after a 30-s contraction, the EDL of alpha2(R/R) mice transported 70.0 nmol/g.min more (86)Rb than WT. Acute sequestration of endogenous ligand(s) in WT mice infused with Digibind to sequester endogenous cardiac glycoside(s) produced similar effects on both resting and contraction-induced (86)Rb transport. Additionally, the alpha2(R/R) mice exhibit an enhanced ability to perform physical exercise, showing a 2.1- to 2.8-fold lower failure rate than WT within minutes of the onset of moderate-intensity treadmill running. Their enhanced exercise performance is consistent with their enhanced contraction-induced Na,K-ATPase transport in the skeletal muscles. These results demonstrate that the Na,K-ATPase alpha2 isozyme in skeletal muscle is regulated dynamically by a mechanism that utilizes the cardiac glycoside-binding site and an endogenous ligand(s) and that its cardiac glycoside-binding site can play a physiological role in the dynamic adaptations to exercise.

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo.

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells.

Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides. K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level. The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex.

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance.

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.

The alpha(1)- and alpha(2)-isoforms of Na-K-ATPase play different roles in skeletal muscle contractility.

The Na-K-ATPase, which maintains the Na(+) and K(+) gradients across the plasma membrane, can play a major role in modulation of skeletal muscle contractility. Although both alpha(1)- and alpha(2)-isoforms of the Na-K-ATPase are expressed in skeletal muscle, the physiological significance of these isoforms in contractility is not known. Evaluation of the contractile parameters of mouse extensor digitorum longus (EDL) was carried out using gene-targeted mice lacking one copy of either the alpha(1)- or alpha(2)-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the alpha(1)- or alpha(2)-isoform, respectively, which permits differentiation of the functional roles of these isoforms. EDL from the alpha(1)(+/-) mouse shows lower force compared with wild type, whereas that from the alpha(2)(+/-) mouse shows greater force. The different functional roles of these two isoforms are further demonstrated because inhibition of the alpha(2)-isoform with ouabain increases contractility of alpha(1)(+/-) EDL. These results demonstrate that the Na-K-ATPase alpha(1)- and alpha(2)-isoforms may play different roles in skeletal muscle contraction.

Lung Krüppel-like factor contains an autoinhibitory domain that regulates its transcriptional activation by binding WWP1, an E3 ubiquitin ligase.

Lung Krüppel-like factor (LKLF/Krüppel-like factor 2), a member of the Krüppel-like factor family of transcription factors, is expressed predominantly in the lungs, with low levels of expression in other organs such as heart, spleen, skeletal muscle, and testis. LKLF is essential during pulmonary development and single-positive T-cell development and is indispensable during mouse embryogenesis. In this study, we performed a series of experiments to define the activation domain of LKLF as a means to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this transcription factor. Using deletion analysis, it is shown that LKLF contains a transcriptional activation domain as well as a strong autoinhibitory subdomain. The inhibitory subdomain is able to independently suppress transcriptional activation of other strong activators such as viral protein 16, VP16. This occurs either when the inhibitory domain is fused directly to VP16 or when the inhibitory domain is independently bound to DNA by GAL4 DNA-binding domain independent of the VP16 activator. Overexpression of the LKLF autoinhibitory domain alone potentiates transactivation by wild type LKLF, suggesting that the inhibitory domain binds a cofactor that prevents LKLF from transactivating. A yeast-two hybrid screen identified WWP1, an E3 ubiquitin ligase that binds specifically to the LKLF inhibitory domain but not to other transcription factors. In mammalian cells, WWP1 functions as a cofactor by binding LKLF and suppressing transactivation. These data demonstrate that LKLF contains multiple domains that either potentiate or inhibit the ability of this factor to function as an activator of transcription; moreover, regulation of LKLF transactivation is attenuated by an E3 ubiquitin ligase, WWP1.

cDNA cloning, mapping and expression of the mouse propionyl CoA carboxylase beta (pccb), the gene for human type II propionic acidaemia.

Propionyl CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of amino acids, odd-chained fatty acids and other metabolites. PCC is composed of two equal subunits, alpha and beta, which are encoded by two separate genes at two distinct human loci. Mutations of either gene in humans results in propionic acidemia (PA). To identify the mouse cDNA for the propionyl CoA carboxylase beta-subunit (pccb), we have screened the mouse EST database using the human sequence. The murine mRNA transcript is approximately 2.3 kb, nearly 500 bps larger than the human approximately 1.8 kb transcript. A PAC genomic DNA clone from the mouse was also isolated and used to generate probes and PCR primers for mapping the pccb locus in the mouse. Both the C57Bl/6JEi and Spret/Ei alleles for regions flanking the pccb gene were sequenced to identify RFLPs. The Jackson Laboratory BSS and BSB backcross panel DNAs were then analyzed using a DdeI polymorphism placing the pccb locus on mouse chromosome 9. Northern blots of adult tissue show that the pccb gene is broadly expressed in the mouse. The approximately 2.3 kb transcript is most abundantly expressed in the kidney, liver, small intestine and stomach tissues.

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala.

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.

Sperm motility is dependent on a unique isoform of the Na,K-ATPase.

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, alpha and beta, and is responsible for translocating Na(+) out of the cell and K(+) into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the alpha and beta subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The alpha4 isoform is the most recently identified Na, K-ATPase alpha isoform, and its expression has been found only in testis. Here we show that expression of the alpha4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the alpha4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.

The GATA-E box-GATA motif in the EKLF promoter is required for in vivo expression.

The erythroid Krüppel-like factor (EKLF) is a key regulatory protein in globin gene expression. This zinc finger transcription factor is required for expression of the adult beta globin gene, and it has been suggested that it plays an important role in the developmental switch from fetal gamma to adult beta globin gene expression. We have previously described a sequence element in the distal promoter region of the mouse EKLF gene that is critical for the expression of this transcription factor. The element consists of an E box motif flanked by 2 GATA-1 binding sites. Here we demonstrate that mutation of the E box or the GATA-1 consensus sequences eliminates expression from the EKLF promoter in transgenic mice. These results confirm the importance of this activator element for in vivo expression of the EKLF gene. (Blood. 2000;95:1652-1655)

Characterization of the lung Krüppel-like transcription factor gene and upstream regulatory elements.

We previously described the isolation and characterization of the cDNA for lung Krüppel-like factor (LKLF), a zinc finger transcription factor that is predominately expressed in the lung of adult mice. In this study, we report the complete structure and nucleotide sequence of the mouse LKLF gene, which is comprised of three exons and two small introns. Moreover, the identification of critical sequence elements required for expression is described using reporter constructs with the LKLF promoter transfected into LA-4 lung cells. Results from these constructs reveal an important region for transcriptional activity that lies between the -490/-72bp upstream sequence. This region contains two canonical Sp1 binding sites that affect expression levels in a non tissue-specific manner. In addition, using a base-pair mutagenesis strategy, a region from -157/-72bp was found to be necessary for upregulating expression. In transfection assays, mutations of the -138/-111bp region resulted in approximately 70-80% loss of promoter activity. This cis-element does not appear to correspond to any known transcription factor consensus sequence. Moreover, mutations within this cis-region disrupt the binding of a protein complex from nuclear extracts of various tissues.

cDNA isolation, genomic structure, regulation, and chromosomal localization of human lung Kruppel-like factor.

Lung Kruppel-like factor (LKLF) is a zinc finger transcription factor critical for embryonic development. We have previously identified and isolated the mouse LKLF gene and examined its role using gene targeting. In this report, we describe the isolation and molecular characterization of the human homolog of murine LKLF. The human and mouse LKLF homologs exhibit an 85% nucleotide identity and share 90% amino acid similarity. Furthermore, the 5' sequence in the proximal promoter region and 3' untranslated region are also conserved between the two species. Of particular interest is the finding that while sequences in the proximal promoter have diverged between mouse and human, a region of 75 nucleotides is essentially identical. Site-directed mutagenesis in this region impairs the ability of the LKLF promoter to drive reporter gene expression, indicating that it represents a novel transcriptional element important in the regulation of LKLF gene expression. The activation domain is highly proline-rich and, similar to mouse LKLF, contains 22% proline residues. The human LKLF transcriptional unit is located in a genomic region of approximately 3 kb on chromosome 19p13.1. This region of chromosome 19 is known to contain genes involved in various human diseases. Like mouse LKLF, human LKLF consists of three exons that are interrupted by two small introns. The locations of intron/exon boundaries and splice sites are conserved between two homologs. Northern analysis shows that LKLF is expressed in lung in addition to heart, skeletal muscle, placenta, and pancreas. The isolation and chromosomal mapping of human LKLF will make it possible to initiate studies devoted to assess the involvement of this gene in human disease(s).

Lung Kruppel-like factor, a zinc finger transcription factor, is essential for normal lung development.

Lung Kruppel-like factor (LKLF) is a member of the Kruppel-like factor family of transcription factors and is highly expressed in lung with limited distribution in other tissues. Mice lacking LKLF due to inactivation of LKLF by gene targeting die in utero at midgestation around day 12.5 due to severe hemorrhage, making it difficult to study the role of this transcription factor in lung development and function. However, in vitro organ culture of lung buds removed from 11.5-day-old LKLF(-/-) embryos show normal tracheobronchial tree formation. To examine later stages of lung development, the embryonic lethality due to germ line LKLF null mutation was circumvented by constructing LKLF homozygous null mouse embryonic stem cells, using a two-step gene targeting procedure, and determining whether these cells give rise to lung tissue. The targeted cells were used to produce chimeric animals, and the contribution of LKLF-deficient cells to the formation of various internal organs was analyzed. In chimeric mice that survived after birth, null embryonic stem cells contributed significantly to all of the major organs except the lungs. On the other hand, some highly chimeric animals died at birth, and histopathological examination of their lungs suggested abnormalities in their lung development. These studies show that LKLF plays an important role in normal lung development.

Alanine scanning mutagenesis of oxygen-containing amino acids in the transmembrane region of the Na,K-ATPase.

Oxygen-containing amino acids in the transmembrane region of the Na, K-ATPase alpha subunit were studied to identify residues involved in Na+ and/or K+ coordination by the enzyme. Conserved residues located in the polar face of transmembrane helices were selected using helical wheel and topological models of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha1 isoform and expressed in HeLa cells. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of heterologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, Ser140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu818, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those carrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were introduced into the wild-type alpha1 sheep isoform and expressed in mouse NIH3T3 cells where [3H]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. Expression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at the plasma membrane was significantly lower than that of the wild-type isoform. Thus, these substitutions appear to impair the maturation process or targeting of the enzyme to the plasma membrane, but not cation-enzyme interactions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-containing residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme function. Consequently, other chemical groups, aromatic rings or backbone carbonyls, should be considered in models of cation-binding sites.

Identification of a specific role for the Na,K-ATPase alpha 2 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.

Characterization of the fourth alpha isoform of the Na,K-ATPase.

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, alpha and beta, and tissue-specific isoforms exist for each of these, alpha1, alpha2 and alpha3 and beta1, beta2 and beta3. We have proposed that an additional alpha isoform, alpha4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative alpha4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in alpha4 isoform cDNA transfected 3T3 cells. Using an alpha4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical KD values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.

Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase alpha subunit.

The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, K-ATPase alpha subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+ and K+ affinities (K1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na+ acting at the extracellular space (Argüello et al. J. Biol. Chem. 271, 24610-24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzymes, while their activation by K+ did not appear affected. Although the Na+ K1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, nHill = 0.75; Tyr771Ser, nHill = 0.92; Control, nHill = 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+ dependence (nHill = 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 left and right arrow E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na+ binding and occlusion without participating in K+-enzyme interactions.

A gene encoding an intestinal-enriched member of the Krüppel-like factor family expressed in intestinal epithelial cells.

The Krüppel-like factors make up a multigene family of transcription factors that have discrete patterns of expression, implying they play important biological roles in the tissues in which they are expressed. We have identified and characterized the cDNA for a novel murine transcription factor that is an additional member of the Krüppel-like family of transcription factors, named intestinal-enriched Krüppel-like factor (IKLF). This gene appears to be a homolog of the human BTEB-2 gene, although it exhibits a different pattern of tissue expression and the translated product is larger. IKLF is expressed in a limited number of tissues; the highest levels of IKLF expression are found in the digestive tract. IKLF shows temporal changes in expression during embryogenesis indicating that this gene is developmentally regulated. In addition, IKLF expression is limited to the epithelial lining of the intestine and is localized primarily to the base of the crypts in the adult intestine. The IKLF cDNA encodes for a 446 amino acid protein and is able to transactivate by binding specific DNA elements that are also recognized by other members of the Krüppel-like family. In addition, mutations in the activation domain attenuate the ability of this protein to function as a transcription factor. Collectively, these findings show that we have identified a transcription factor that is expressed predominantly in the epithelial crypt cells of the gastrointestinal tract and is a member of the Krüppel-like family of transcription factors.

Loss of LKLF function results in embryonic lethality in mice.

Lung Kruppel-like factor (LKLF) is a member of the Kruppel-like family of zinc finger transcription factors and is closely related to erythroid kruppel-like factor (EKLF), which is necessary for beta-globin gene expression. While EKLF is expressed exclusively in erythroid cells, LKLF is expressed temporally during early embryonic development and predominantly in the adult mouse lung. To understand the role this novel transcription factor plays in development as well as tissue differentiation and function, animals lacking LKLF were produced using gene targeting technology. Mice lacking LKLF die in utero between day 11.5 and 13.5 of embryonic life and exhibit retarded growth, craniofacial abnormalities, abdominal bleeding and signs of anaemia. Although the yolk sac erythropoiesis is normal in mutant embryos, in vitro fetal liver cultures of these embryos fail to give rise to erythroid cells. Expression of other erythroid specific genes such as EKLF, GATA1 and GATA3 is unaltered in these animals. These findings demonstrate the LKLF function is indispensable during normal embryonic development, and although both LKLF and EKLF recognize common DNA motifs, they do not substitute for each other.