Tomás A. Santa-Coloma

Interleukin-1β regulates CFTR expression in human intestinal T84 cells

Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.

Anp32e (Cpd1) and related protein phosphatase 2 inhibitors.

Mouse Anp32e (Acidic leucine-rich nuclear phosphoprotein 32 family, member e: NM_023210, P97822, formerly Cpd1), a protein identified in postnatal cerebellum by differential display, belongs to the superfamily of leucine rich repeat (LRR) proteins and to the Acidic Nuclear Phosphoprotein 32 (ANP32) family of protein phosphatase 2 (PPP2, formerly PP2A) inhibitors. Two families of PPP2 inhibitor proteins have been described, ANP32 and SET, represented by the human proteins ANP32A (NM_006305, formerly LANP, PP32, I1PPP2, PHAPI, MAPM, mapmodulin) and SET (NM_003011, formerly PHAPII, 2PPP2, I2PPP2, TAF-1BETA). Besides their common PPP2 inhibitor activity, described several years ago, these nucleo-cytoplasmic shuttling phosphoproteins have additional and very important functions recently reported. In HeLa cells, ANP32A, SET (isoforms A and B) and ANP32B (APRIL), form a multi-subunit heterocomplex with ELAVL1 (NM_001419, formerly HuR), a protein that stabilizes short-lived mRNAs containing AU-rich elements (AREs). A similar heterocomplex, formed by SET (A and B) and ANP32A as major subunits, possess histone acetyltransferase inhibitory activity (INHAT), and have a role in chromatin remodeling and transcriptional regulation (histone code). The possible roles of these multifunctional proteins are discussed here, with emphasis on mouse Anp32e and the cerebellar tissue.

Tyrosine Kinase c-Src Constitutes a Bridge between Cystic Fibrosis Transmembrane Regulator Channel Failure and MUC1 Overexpression in Cystic Fibrosis

Cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) chloride channel, is associated in the respiratory system with the accumulation of mucus and impaired lung function. The role of the CFTR channel in the regulation of the intracellular pathways that determine the overexpression of mucin genes is unknown. Using differential display, we have observed the differential expression of several mRNAs that may correspond to putative CFTR-dependent genes. One of these mRNAs was further characterized, and it corresponds to the tyrosine kinase c-Src. Additional results suggest that c-Src is a central element in the pathway connecting the CFTR channel with MUC1 overexpression and that the overexpression of mucins is a primary response to CFTR malfunction in cystic fibrosis, which occurs even in the absence of bacterial infection.

CFTR activity and mitochondrial function.

Cystic Fibrosis (CF) is a frequent and lethal autosomal recessive disease, caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Before the discovery of the CFTR gene, several hypotheses attempted to explain the etiology of this disease, including the possible role of a chloride channel, diverse alterations in mitochondrial functions, the overexpression of the lysosomal enzyme α-glucosidase and a deficiency in the cytosolic enzyme glucose 6-phosphate dehydrogenase. Because of the diverse mitochondrial changes found, some authors proposed that the affected gene should codify for a mitochondrial protein. Later, the CFTR cloning and the demonstration of its chloride channel activity turned the mitochondrial, lysosomal and cytosolic hypotheses obsolete. However, in recent years, using new approaches, several investigators reported similar or new alterations of mitochondrial functions in Cystic Fibrosis, thus rediscovering a possible role of mitochondria in this disease. Here, we review these CFTR-driven mitochondrial defects, including differential gene expression, alterations in oxidative phosphorylation, calcium homeostasis, oxidative stress, apoptosis and innate immune response, which might explain some characteristics of the complex CF phenotype and reveals potential new targets for therapy.

Intracellular calcium variation in heparin-capacitated bovine sperm.

This report investigates the mechanisms by which heparin induces capacitation of sperm. Capacitation was determined with chlortetracycline and intracellular calcium concentration, [Ca2+]i, with FURA 2‐AM. Atter 15 minutes incubation with heparin, [Ca2+]i was increased 60% over basal, reaching a plateau thereafter. Sixty percent of calcium entry was inhibited by methoxy‐verapamil, suggesting that activation of voltage dependent calcium channels (VDCC) may be involved in the process. A significant correlation (R2 = 0.88, p<0.006) was found between the percentage of capacitated spermatozoa and the [Ca2+]i increase. The effects of heparin on both processes were blocked by the protein kinase C (PKC) inhibitors staurosporine (100%) and GF‐109203X (90%). It is concluded that heparin may induce sperm capacitation and calcium influx mainly through VDCC (approx. 60%), and also through other membrane systems (40%). Both systems of calcium entry as well as the capacitation process appear to involve on PKC activity.

Early effects of insulin-like growth factor-1 in activated human T lymphocytes

This study evaluates the effects of insulin‐like growth factor (IGF)‐1 receptor (IGF‐1R) down‐regulation in stimulated T lymphocytes by investigating the expression of early activation proteins CD69, CD25, and interleukin (IL)‐2. We found that IGF‐1 does not modify CD69 expression but increases transcription and protein synthesis of CD25 and IL‐2. The lowest level of IGF‐1R detected after 15 min of activation suggested that the effects of IGF‐1 occur at the initiation of cell activation. The activation of IGF‐1R was confirmed by IGF‐1R phosphorylation and increased phosphorylation of microtubule‐associated protein kinase. We also detected the alternative IGF‐1 transcripts Ea, with paracrine/autocrine regulation, and Eb, with endocrine regulation, in Jurkat cells and in quiescent T lymphocytes, and we detected IGF‐1 protein in the culture medium after stimulation. These data suggest that the proliferative effects of IGF‐1 on T lymphocytes include both autocrine/paracrine and endocrine processes.

Differential expression of CPD1 during postnatal development in the mouse cerebellum

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.

A synthetic peptide encompassing two discontinuous regions of hFSH-β subunit mimics the receptor binding surface of the hormone

Synthetic peptides corresponding to discontinuous segments of the hFSH-beta subunit, amino acids 33-53 and 81-95, have been shown to interact with the follicle-stimulating hormone (FSH) receptor. In this study, we demonstrate that hFSH-beta-(33-53)-(81-95)-peptide amide, a synthetic peptide encompassing these binding regions, possesses higher affinity for the FSH receptor than either synthetic hFSH-beta-(33-53) or hFSH-beta-(81-95). This increased affinity suggests that each binding component is effectively interacting with the receptor, providing evidence that these two separate receptor binding regions of hFSH-beta form a continuous binding surface on the native molecule. These results also suggest that binding surfaces of very complex proteins, such as the heterodimeric glycoprotein hormone FSH, may be mimicked by a linear arrangement of its binding domains. A model based on energetics of the peptide-receptor interaction is also described. The results indicate that the affinity (Ka) of a peptide containing different binding domains can be approximated utilizing the product of the affinity constant of each binding domain (Ka = k1.k2...kn).

The Mitochondrial Complex I Activity Is Reduced in Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function

Cystic fibrosis (CF) is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I). Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA) of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines) compared with CFDE/6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR). Moreover, colon carcinoma T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh)-172 or GlyH101) or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells.

Structure-function relationships of the glycoprotein hormones and their receptors

The primary structures of the glycoprotein hormones follitropin (FSH), lutropin (LH), human choriogonadotropin (hCG) and thyrotropin (TSH) have been determined, hCG has been crystallized and initial diffraction data obtained. Studies with synthetic peptides have provided information on regions involved in receptor interaction and signal transduction. The receptors for the glycoprotein hormones have been prepared by gene cloning methods and their primary structures deduced. As Leo Reichert and colleagues discuss here, although cAMP is involved in glycoprotein hormone signal transduction, recent evidence also implicates other second messengers, especially Ca2+ and may include both the phosphatidylinositol pathway and activation of Ca2+ channels.