Functional vasoactive intestinal polypeptide (VIP) receptors in human neuroblastoma subclones that contain VIP precursor mRNA and release VIP-like substances

Three phenotypically distinct subclones (SH-SY-5Y, SH-EP, SH-IN) of the human neuroblastoma cell line SK-N-SH were found to possess vasoactive intestinal polypeptide (VIP) precursor mRNA, release immunoreactive VIP, and express high-affinity VIP receptors coupled to adenylate cyclase. The apparent molecular mass for the receptor polypeptide, as determined by covalent cross-linking of 125I-VIP, was 49 kDa. After 2 days in culture, a concentration of immunoreactive VIP equivalent to the binding affinity of VIP to its receptor was found in the medium in two of these clones (SH-IN and SH-EP). Conditioned medium from SH-IN cells competitively displaced 125I-VIP binding and increased cAMP levels in SH-EP cells, indicating that all of the necessary components for a potential autocrine action of VIP exist in SK-N-SH cells. After numerous cell passages, the SH-EP subclone converted to a distinct phenotype in which VIP precursor mRNA and VIP immunoreactivity in the cell and medium were no longer detectable. In correlation, the VIP receptor number increased, and the EC50 for VIP stimulation of cAMP production shifted to a lower concentration. This points to the possibility that the continuous presence of endogenous VIP in earlier passage SH-EP cells causes a modification in VIP receptor number and cell responsiveness to VIP.     

The Epithelial Phenotype of Human Neuroblastoma Cells Expresses Bradykinin, Endothelin, and Angiotensin II Receptors That Stimulate Phosphoinositide Hydrolysis

The neuroblastoma line SK-N-SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH-SY5Y), an epithelial phenotype (SH-EP), and an intermediate cell type (SH-IN). In SH-SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH-EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of bradykinin greater than endothelin greater than angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH-EP cells but not in SH-SY5Y cells. In the intermediate cell clone, SH-IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides--bradykinin, endothelin, and angiotensin II--on phosphoinositide hydrolysis in SH-EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH-EP cells with pertussis toxin under conditions sufficient to ADP-ribosylate 90-95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short-term exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (1 microM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH-EP cells and partially inhibited that by muscarinic receptors in SH-SY5Y cells. Prolonged incubation of SH-EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH-SY5Y cells. The pattern of phorbol ester-mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH-IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH-EP than to muscarinic receptors in SH-SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.     

Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells

The tumor promoter phorbol 13-myristate 12-acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP-1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase-2 (PGHS-2) expression in murine osteoblastic MC3T3-E1 cells, but no functional AP-1 binding motifs in the 5'-flanking region have been identified. In MC3T3-E1 cells transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS-2 promoter region identified three potential AP-1 elements in the -371/+70 bp region, and deletion analysis suggested that the sequence 5'-aGAGTCA-3' at -69/-63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP-1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS-2 probe spanning this sequence, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Mutation of this AP-1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3-E1 cells. On EMSA, serum induces binding to a PGHS-2 probe spanning the AP-1 site, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Joint mutation of this AP-1 site and the nearby CRE site at -56/-52 bp, previously shown to mediate serum, v-src and PDGF induction of PGHS-2 in NIH-3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3-E1 cells. Hence, the AP-1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS-2 promoter activity.     

cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester

The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion. Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined. The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells. PMA treatment had no effect on luciferase activity in Calu-3 cells transiently transfected with plasmids containing luc driven by up to 2.3 kb of CFTR 5'-flanking DNA. PMA increased luciferase activity in transfected HT-29 cells. A more extensive region of DNA was evaluated using a yeast artificial chromosome (YAC) containing luc driven by approximately 335 of CFTR 5'-flanking DNA (y5'luc) stably introduced into HT-29 cells. Clonal cell lines containing y5'luc were created and assessed for luciferase activity at baseline and in response to forskolin and PMA. There was a wide range of baseline luciferase activities among the clones (42-1038 units/microg protein) that was not entirely due to the number of luc copies present within the cells. Treatment with both PMA and forskolin led to increased luciferase activity in six randomly selected clonal cell lines. As expected, endogenous CFTR expression increased in response to forskolin and decreased in response to PMA. These studies demonstrate that luc-containing YAC vectors can be used to study CFTR expression in human cells. In addition, these data suggest that important regulatory elements responsible for decreased CFTR expression in response to PMA are not located upstream of CFTR in the approximately 335 kb 5'-flanking sequence included in this YAC construct.     

Leukoregulin, a T-cell derived cytokine, upregulates stromelysin-1 gene expression in human dermal fibroblasts: evidence for the role of AP-1 in transcriptional activation.

Leukoregulin (LR), a product of activated T-cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455-1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin-1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose- and time-dependent elevation of stromelysin-1 mRNA levels, the maximum enhancement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 1.3 kb of 5' flanking DNA of the human stromelysin-1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This enhancement was abolished by a single base substitution in the AP-1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP-1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin-1 gene expression by LR is mediated by AP-1 regulatory elements which are necessary, but not sufficient, for gene response.