A mammalian arginine/lysine transporter uses multiple promoters.

The mCAT-2 gene encodes a Na(+)-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.

A 20-nucleotide element in the intestinal fatty acid binding protein gene modulates its cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of expression in transgenic mice.

A sequence of epithelial cell proliferation, allocation to four principal lineages, migration-associated differentiation, and cell loss occurs along the crypt-villus axis of the mouse intestine. The sequence is completed in a few days and is recapitulated throughout the life-span of the animal. We have used an intestine-specific fatty acid binding protein gene, Fabpi, as a model for studying regulation of gene expression in this unique developmental system. Promoter mapping studies in transgenic mice identified a 20-bp cis-acting element (5'-AGGTGGAAGCCATCACACTT-3') that binds small intestinal nuclear proteins and participates in the control of Fabpi's cephalocaudal, differentiation-dependent, and cell lineage-specific patterns of expression. Immunocytochemical studies using confocal and electron microscopy indicate that it does so by acting as a suppressor of gene expression in the distal small intestine/colon, as a suppressor of gene activation in proliferating and nonproliferating cells located in the crypts of Lieberkühn, and as a suppressor of expression in the growth factor and defensin-producing Paneth cell lineage. The 20-bp domain has no obvious sequence similarities to known transcription factor binding sites. The three functions modulated by this compact element represent the types of functions required to establish and maintain the intestine's remarkably complex spatial patterns of gene expression. The transgenes described in this report also appear to be useful in characterizing the crypt's stem cell hierarchy.

A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice.

A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.

Three-dimensional scroll waves of cAMP could direct cell movement and gene expression in Dictyostelium slugs.

Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.

bcl-2 transgene expression can protect neurons against developmental and induced cell death.

The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

Eph/Ephrin memhrane proteins: A mammalian expression vector pTIg-BOS-Fc allowing rapid protein purification

There is an urgent need for high purity, single chain, fully functional Eph/ephrin membrane proteins. This report outlines the pTIg-BOS-Fc vector and purification approach resulting in rapid increased production of fully functional single chain extracellular proteins that were isolated with high purity and used in structure-function analysis and pre-clinical studies.

Expression and role of complex carbohydrates in axon guidance in the olfactory system

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood groups Sda (or CT1 antigen) and H are expressed by primary sensory neurons in the olfactory system, while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons only in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the blood group H and A carbohydrates were not expressed in the olfactory systems which caused delayed development of the nerve fibre and glomerular layers in the main olfactory bulb. In contrast, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice perturbed the ability of vomeronasal axons to terminate in the accessory olfactory bulb and affected the selective targeting of axons in the main olfactory bulb. During regeneration following bulbectomy, vomeronasal axons were unable to effectively sort out from the main olfactory axons when blood group A was misexpressed. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development and regeneration of the olfactory nerve pathways.

PAX2 activates WNT4 expression during mammalian kidney development

The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the S-shaped body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5'-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.

Comparative gene expression analysis of NKT cell subpopulations

Natural killer T (NKT) cells are a lymphocyte lineage, which has diverse immune regulatory activities in many disease settings. Most previous studies have investigated the functions of this family of cells as a single entity, but more recent evidence highlights the distinct functional and phenotypic properties of NKT cell subpopulations. It is likely that the diverse functions of NKT cells are regulated and coordinated by these different NKT subsets. Little is known about how NKT subsets differ in their interactions with the host. We have undertaken the first microarray analysis comparing the gene expression profiles of activated human NKT cell subpopulations, including CD8(+) NKT cells, which have often been overlooked. We describe the significant gene expression differences among NKT cell subpopulations and some of the molecules likely to confer their distinct functional roles. Several genes not associated previously with NKT cells were shown to be expressed differentially in specific NKT cell subpopulations. Our findings provide new insights into the NKT cell family, which may direct further research toward better manipulation of NKT cells for therapeutic applications.

Cell surface expression of urokinase receptor in normal mammary epithelial cells and breast cancer cell lines

Background: The urokinase receptor (uPAR) is important in the process of extracellular matrix degradation occurring during cancer cell invasion and metastasis. We wished to quantify uPAR on the surfaces of normal mammary epithelial cells (HMEC) and 6 well-known breast cancer cell lines using flow cytometry. Materials and Methods: Cell surface uPAR was labelled with a monoclonal antibody, and this was detected with a florescent-labelled second antibody and accurately measured using flow cytometry. The measured fluorescent signals of the stained cells were interpolated with those of Quantum Simply Cellular bead standards to determine the number of uPAR sites per cell. Results: The breast cancer cell lines ranged from 13,700 to 50,800 uPAR sites per cell, whilst HMEC cells had only 2,500 sites. Conclusions: This simple and reliable method showed that the expression of cell surface uPAR is higher in the breast cancer cell lines than in the normal mammary cells.

Expression of apoptotic regulatory molecules in renal cell carcinoma: Elevated expression of Fas ligand

Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO- 1/CD95), Fas ligand (Fas L) and bcl-2 in these tumours. The expression of Fas, Fas L and bcl-2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio-labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde-3-phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl-2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down-regulated in the patients' versus normal peripheral blood (P = 0.026). Most interestingly, significantly up-regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl-2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated.

Electrolyte transport in the mammalian colon: mechanisms and implications for disease Kunzelmann and Marcus Mall

Electrolyte Transport in the Mammalian Colon: Mechanisms and Implications for Disease. Physiol. Rev. 82: 245-289, 2002.The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na+ channels (ENaC), the Na+-K+-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na+ feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl- secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl- channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl- secretion and enhanced Na+ absorption in the colon of cystic fibrosis (CF) patients. Ca2+- and cAMP-activated basolateral K+ channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.