Human interleukin 4 receptor confers biological responsiveness and defines a novel receptor superfamily.

IL-4, a pleiotropic cytokine produced by T lymphocytes, plays an important role in immune responsiveness by regulating proliferation and differentiation of a variety of lymphoid and myeloid cells via binding to high affinity receptors. In this report we describe the isolation and functional expression of a human IL-4-R cDNA. When transfected into COS-7 cells, the cDNA encodes a 140-kD cell-surface protein. After transfection into a murine T cell line, the cDNA encodes a protein that binds human IL-4 with high affinity and can confer responsiveness to human IL-4. The predicted extracellular domain of the IL-4-R exhibits significant amino acid sequence homology with the beta subunit of the IL-2-R (p75), and the receptors for IL-6, erythropoietin, and prolactin. These receptors comprise a novel superfamily with extracellular domains characterized by four conserved cysteine residues and a double tryptophan-serine (WSXWS) motif located proximal to the transmembrane region.

A chicken transferrin gene in transgenic mice escapes X-chromosome inactivation.

Mammalian X-chromosome inactivation involves a coordinate shutting down of physically linked genes. Several proposed models require the presence of specific sequences near genes to permit the spread of inactivation into these regions. If such models are correct, one might predict that heterologous genes transferred onto the X chromosome might lack the appropriate signal sequences and therefore escape inactivation. To determine whether a foreign gene inserted into the X chromosome is subject to inactivation, transgenic mice harboring 11 copies of the complete, 17-kilobase chicken transferrin gene on the X chromosome were used. Male mice hemizygous for this insert were bred with females bearing Searle's translocation, an X-chromosome rearrangement that is always active in heterozygous females (the unrearranged X chromosome is inactive). Female offspring bearing the Searle's translocation and the chicken transferrin gene had the same amount of chicken transferrin messenger RNA in liver as did transgenic male mice or transgenic female mice lacking the Searle's chromosome. This result shows that the inserted gene is not subject to X-chromosome inactivation and suggests that the inactivation process cannot spread over 187 kilobases of DNA in the absence of specific signal sequences required for inactivation.

A p53-dependent mouse spindle checkpoint.

Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.

A new cytokine receptor superfamily.

The amino acid sequences of several, recently cloned cytokine receptors show significant homologies, primarily in their extracellular, ligand-binding domains. With one exception, their cognate cytokines mediate biological activities on a variety of hematopoietic cell types; thus we have designated the receptors as the hematopoietic receptor superfamily.

Reproductive function in protein kinase inhibitor-deficient mice.

The protein kinase inhibitor (PKI) family includes three genes encoding small, heat-stable inhibitors of the cyclic AMP-dependent kinase PKA. Each PKI isoform contains a PKA inhibitory domain and a nuclear export domain, enabling PKI to both inhibit PKA and remove it from the nucleus. The PKIbeta isoform, also known as testis PKI, is highly expressed in germ cells of the testis and is found at more modest levels in other tissues. In order to investigate its physiological role, we have generated PKIbeta knockout mice by gene targeting. These mice exhibit a partial loss of PKI activity in testis but remain fertile with normal testis development and function. PKIbeta knockout females also reproduce normally. The PKIbeta mutants were crossed with our previously derived PKIalpha mutants to obtain double-knockout mice. Remarkably, these mice are also viable and fertile with no obvious physiological defects in either males or females.

Estrogen regulation of the avian transferrin gene in transgenic mice.

The intact chicken transferrin gene was microinjected into fertilized mouse eggs, and the resulting transgenic animals were used to produce lines of mice containing integrated copies of the chicken gene. The levels of expression of the chicken gene were quantitated in various tissues, and the response of the gene to estrogen stimulation was measured after chronic or acute estrogen exposure. Two of the three mouse lines studied maintained stable levels of expression in successive generations of offspring, and the third line had two- to threefold-higher levels in offspring than in the original parent. In the third line, the original transgenic parent was found to be a mosaic. The chicken transferrin gene was expressed at 10- to 20-fold-higher levels in liver than in any other tissue; however, the levels of chicken transferrin mRNA in kidney were higher than expected, indicating that the tissue specificity was only partial. In all three lines, the foreign gene was induced by estrogen administration. After 10 days of estrogen administration, there was a twofold increase in both transferrin mRNA and transcription of the chicken transferrin gene. A single injection of estradiol led to a fourfold increase in transferrin mRNA synthesis at 4h. As a control the levels of mouse albumin were measured, and both the level of albumin mRNA and its rate of transcription declined about twofold after estrogen administration. Our results indicate that the intact chicken gene with 2.2 kilobases of 5' flanking sequence contains signals for both tissue specificity and steroid regulation that can be recognized in mice.

Expression from the transferrin gene promoter in transgenic mice.

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and S1 nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3- to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four- to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.

Deficient gene expression in protein kinase inhibitor alpha Null mutant mice.

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

PKA isoforms, neural pathways, and behaviour: making the connection.

In mammals, the cAMP-dependent protein kinase (PKA) family of enzymes is assembled from the products of four regulatory and two catalytic subunit genes, all of which are expressed in neurons. Specific isoforms of PKA display differences in biochemical properties and subcellular localization, but it has been difficult to ascribe specific physiological functions to any given isoform. The recent development of gene knockout and transgenic mouse models has allowed for a more integrated examination of the in vivo roles of specific PKA isoforms in gene expression, synaptic plasticity, and behaviour.

Directed expression of an oncogene to Sertoli cells in transgenic mice using mullerian inhibiting substance regulatory sequences.

Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.

Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells.

Transferrin-specific cDNA clones were isolated from a rat liver cDNA library prepared from transferrin-enriched mRNA. Hybrid selection and sequence analysis confirmed that the selected clone contained the carboxy-terminal coding region of the transferrin mRNA. Northern blot analysis was used to demonstrate the presence of transferrin mRNA in liver and Sertoli cells. Transferrin mRNA levels were measured in total RNA isolated from cultured rat Sertoli cells after treatment with FSH, insulin, retinol, and testosterone. The results showed a 2- to 4-fold increase in the level of transferrin mRNA, which peaked on the fourth day of culture after initiation of treatment, with FSH, insulin, retinol, and testosterone. This induction is gene specific, since no change in the mRNA levels for either the catalytic or regulatory subunits of cAMP-dependent protein kinase was observed. The effects of hormones, vitamin A (retinol), and Bu2 cAMP on transferrin mRNA and transferrin secretion (measured by RIA) in cultured Sertoli cells were compared. In general, a direct relationship between the amount of transferrin mRNA present in the cells and the amount of transferrin secreted into the culture medium was observed. These results demonstrate the important role that vitamin A, testosterone, and peptide hormones play in modulating transferrin gene expression in Sertoli cells.

Expression of receptors for interleukin 4 and interleukin 7 on human T cells.

Human recombinant interleukin 4 (IL-4) and interleukin 7 (IL-7) have been modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 and IL-7 receptors (R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA) or immobilized CD3 monoclonal antibody the intensity of biotinylated IL-4 staining increased approximately twofold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused a considerable increase in IL-4 receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R although it was able to cause a significant increase in receptor expression on T cells co-cultured with PHA or CD3. Freshly isolated T cells expressed high levels of IL-7R, as determined by biotinylated IL-7 binding and flow cytometry, which did not change significantly with culture in medium alone. Stimulation with PHA, Concanavalin A (Con A) or CD3 had little effect on the intensity of staining. In contrast, activation with phorbol ester resulted in a decrease in IL-7R expression. Similarly, in the presence of IL-4 or IL-7, but not IL-2, the intensity of staining with biotinylated IL-7 was lowered. Analysis of purified T-cell populations showed that IL-7R were present, and IL-4R could be induced, on both CD4+ and CD8+ populations. Analysis of IL-4 receptor expression by this flow cytometric technique was supported by results from 125I-labeled IL-4 binding and by Northern blot analysis of mRNA levels. Taken together, the results of these studies show that the use of biotinylated cytokines and flow cytometry provides a very sensitive method with which to study the expression and regulation of cytokine receptors.

CD44 expression results in the synthesis of unusual RNAs and multiple protein species containing core polypeptide sequences.

Several cDNA clones encoding portions of the primate CD44 molecule have been isolated. Nonprototypic clones are moderately abundant in a cDNA library, with four unusual clones identified out of the 25 examined. Two of these clones appear to be the result of incomplete RNA splicing events. The baboon genome appears to contain multiple CD44 related sequences. Cell surface CD44 is expressed in greatly differing amounts in different tissues. These levels are generally paralleled by CD44 mRNA levels. Analysis of the relationship between the 90 kDa and 180-200 kDa with two polypeptide specific antisera indicates that these two antigens share extensive amino acid similarity and are likely to be differentially modified versions of the same gene product.

Rat transferrin gene expression: tissue-specific regulation by iron deficiency.

Rats raised on a low-iron diet were used as a model system for investigating the regulation of transferrin gene expression by iron deficiency. We quantitated transferrin mRNA in a variety of tissues from normal and iron-deficient rats and found that the level of transferrin mRNA in normal rat liver was about 6500 molecules per cell, while the level in iron-deficient animals was 2.4-fold higher. The increase of transferrin mRNA in iron deficiency was the result of a specific induction of transferrin gene transcriptional activity as measured in isolated nuclei. This increase in transferrin gene expression resulted in a corresponding increase in serum total-iron-binding capacity. Of the other tissues examined, moderate amounts of transferrin mRNA were found in brain (83 molecules per cell) and testis (114 molecules per cell), and low levels were measured in spleen and kidney. The transferrin mRNA content of brain, testis, spleen, and kidney remained unchanged in iron deficiency. The small intestine had no detectable transferrin mRNA in either normal or iron-deficient rats; however, transferrin protein was present, and its level was 2-fold higher in the iron-deficient group. We hypothesize that intestinal transferrin is synthesized in the liver and is delivered to the gut via the bile. Consistent with this idea, bile transferrin content was found to be elevated in iron deficiency and appeared to be sufficient to account for intestinal transferrin levels. In addition, treatment of plasma transferrin with bile caused an acidic shift in its isoelectric-focusing behavior so that it comigrated with intestinal transferrin.

Regulation of interleukin 4 receptors on human T cells.

Human recombinant interleukin 4 (IL-4) was modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 receptors (IL-4R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA), or immobilized CD3 mAb the intensity of biotinylated IL-4 staining increased approximately 2-fold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused an increase in receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R, although it was able to induce a significant increase in receptor expression on T cells co-cultured with PHA or CD3 mAb. Flow cytometric analysis of purified T cell subsets confirmed that the up-regulation of IL-4R occurred on both CD4+ and CD8+ subpopulations. Two-color staining of T cells activated with PHA and IL-7 revealed that this increase in IL-4R expression occurred almost exclusively on cells expressing the p55 IL-2Ra subunit, although a significant number of cells expressing p55 do not express IL-4R. Analysis of IL-4R expression by this flow cytometric technique was substantiated by 125I-labeled IL-4 binding data and Northern blot analysis of IL-4R mRNA levels, suggesting that use of biotinylated human IL-4 for ligand binding and its detection by flow cytometry provides a very sensitive method for the study of IL-4R regulation.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.