Heterologous Cell Lines Research Articles

Multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species

Primary cell cultures prepared from embryos of the thrips species Frankliniella occidentalis and Thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (TSWV). Using polyclonal antibodies against the viral nucleocapsid protein (N) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a F. occidentalis, and a T. tabaci population which failed to transmit the virus. The infection was found in approximately 40% of the monolayer cells. Using antibodies against a nonstructural protein (NSs) of TSWV, uniform and more diffused staining was observed throughout the cytoplasm of these cells, underlying active genome replication. The NSs protein accumulated slower than the N protein in the cells of both thrips species. No multiplication of TSWV was observed in a heterologous insect cell line, i.e. from Spodoptera frugiperda, suggesting the existence of specific host factors in the thrips-derived cells.

Feedback modulation of ligand-engaged alpha L/beta 2 leukocyte integrin (LFA-1) by cyclic AMP-dependent protein kinase.

Rapid interconversion between a firmly adherent and a nonadherent, circulating phenotype is a distinctive feature of mature leukocytes and is thought to be essential for efficient immune surveillance. Leukocyte adhesion is a finely regulated process controlled in part by reversible, activation-dependent up-regulation of beta 1- and beta 2- integrin function. To investigate the molecular basis of such reversibility in human T lymphocytes, we developed a model of alpha L/beta 2 (LFA-1)-dependent adhesion that uses a heterologous cell line expressing human intercellular adhesion molecule-1 as a selected ligand. We show here that intracellular cAMP elevation, followed by cAMP-dependent kinase activation, promotes T cell deadhesion by disassembling the actin-based cytoskeleton, thus dissociating LFA-1 from cytoskeletal anchoring proteins that normally connect the adhesion receptor to F-actin in lymphocytes engaged in intercellular adhesion. Cells costimulated via the CD3 and LFA-1 receptors by specific Abs or by binding to intercellular adhesion molecule-1 display gradual and persistent intracellular cAMP elevations due to the synergistic induction of a protein kinase C-dependent adenylyl cyclase isoform. On the basis of these findings, we propose a feedback model for short term regulation of leukocyte integrins, involving sequential, integrin-dependent activation of the protein kinase C and adenylyl cyclase/cAMP-dependent kinase enzymatic pathways and leading to disengagement of the adhesion receptor from its specific ligand.

Thyrotropin (TSH)-releasing hormone stimulates TSH beta promoter activity by two distinct mechanisms involving calcium influx through L type Ca2+ channels and protein kinase C.

TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.

A Dopamine-responsive Domain in the N-terminal Sequence of Pit-1: TRANSCRIPTIONAL INHIBITION IN ENDOCRINE CELL TYPES

The POU transcription factor Pit-1 activates the prolactin gene in pituitary lactotrophs and may integrate responses of the gene to external signals. To study the role of Pit-1 in dopaminergic inhibition of the prolactin gene, we transiently transfected Pit-1 and dopamine D2 receptor vectors into a series of heterologous cell lines and examined dopamine regulation of the prolactin gene promoter. Regulation was Pit-1-dependent in all cell lines tested. Moreover, dopamine responsiveness was cell type-specific: stimulatory in fibroblasts (COS-7) and muscle-type cells (P19/Me2SO-induced) and inhibitory in pancreatic endocrine (RIN, InR1-G9) and neural-like (P19/retinoic acid-induced) cells. Because dopaminergic responses in Pit-1-transfected RIN cells paralleled those in pituitary GH4 cells, the islet cell line was used to test for sequences in Pit-1 that mediate negative hormone signals. Dopamine responsiveness of the Pit-1 transactivation domain (residues 8-80) was examined using a chimeric LexA construct. LxPit-1, LxSp1, and Lx-glucocorticoid receptor fusions all activated basal transcription, but only LxPit-1 was regulated by dopamine. Regulatory responses of LxPit-1 and full-length Pit-1 were quantitatively similar. In addition, gain-of-function G alpha mutants that inhibit Pit-1-dependent promoters in GH4 cells also suppressed selectively Pit-1- or LxPit-1-dependent promoters in RIN cells. This demonstrates that Pit-1 can function as a specific target for distinct inhibitory G protein signals. Interestingly, Pit-1 sequences N-terminal to the DNA-binding POU domain appear to be sufficient in mediating regulation by these pathways.

Functional Reconstitution of the Phagocyte NADPH Oxidase by Transfection of Its Multiple Components in a Heterologous System

The phagocyte NADPH oxidase system, as previously defined by cell-free reconstitution, is comprised of five essential components, three of which are produced during late phagocytic differentiation--namely, two cytosolic proteins, p47- and p67-phox--and the large subunit of cytochrome b558, gp91-phox. To confirm that these are the only phagocyte-specific components necessary for oxidase activity in whole cells, the recombinant NADPH oxidase was reconstituted in a heterologous cell line. An undifferentiated multipotent leukemic cell line, K562, which expresses endogenous Rac and the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected with episomal expression vectors containing cDNAs for the three other oxidase components. After 4 days of selection, the complete oxidase system was functionally reconstituted in transfected cells stimulated with phorbol myristate acetate or calcium ionophore. These easily transfected cells provide an ideal model system in which several oxidase components can be genetically manipulated and readily expressed. This system can be used to test the effects of mutations associated with any of the genes affected in chronic granulomatous disease and will facilitate studies on structure-function relationships within several oxidase components. This system will also aid in delineation of upstream regulators functioning through various signaling pathways.

Immunology and morphology studies on the proliferation of in vitro cultivated Echinococcus multilocularis metacestodes.

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis (AE) in various mammals, including humans. Traditionally metacestodes are maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils. However, in animal models it has always been difficult to draw definite conclusions about the factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexicity of the host-parasite interplay. This paper describes the maintenance and proliferation of E. multilocularis metacestodes as well as the formation of protoscolices in a chemically defined medium devoid of host influence. The interactive role of a heterologous human cell line (CACO2) in the in vitro development of metacestodes was also assessed. The morphology and ultrastructure of in vitro-generated metacestodes was studied using scanning (SEM) and transmission electron microscopy (TEM). Different cultivation procedures were analyzed in terms of expression of B- and T-cell epitopes and of the relevant laminated layer-antigen Em2; the exact localization of this antigen was further demonstrated by immunogold electron microscopy.

Sodium channel mutations in paramyotonia congenita exhibit similar biophysical phenotypes in vitro.

Mutations in the skeletal muscle voltage-gated Na+ channel alpha-subunit have been found in patients with two distinct hereditary disorders of sarcolemmal excitation: hyperkalemic periodic paralysis (HYPP) and paramyotonia congenita (PC). Six of these mutations have been functionally expressed in a heterologous cell line (tsA201 cells) using the recombinant human skeletal muscle Na+ channel alpha-subunit cDNA hSkM1. PC mutants from diverse locations in this subunit (T1313M, L1433R, R1448H, R1448C, A1156T) all exhibit a similar disturbance in channel inactivation characterized by reduced macroscopic rate, accelerated recovery, and altered voltage dependence. PC mutants had no significant abnormality in activation. In contrast, one HYPP mutation studied (T704M) has a normal inactivation rate but exhibits shifts in the midpoints of steady-state activation and inactivation along the voltage axis. These findings help to explain the phenotypic differences between HYPP and PC at the molecular and biophysical level and contribute to our understanding of Na+ channel structure and function.

Differential expression of RNA transcripts encoding unique carboxy-terminal sequences of human parathyroid hormone-related peptide.

The human gene encoding PTH-related peptide (hPTHrP) is a complex transcriptional unit that gives rise to multiple messenger RNA (mRNA) transcripts in normal and neoplastic tissues. The utilization of three different transcription start sites and alternative splicing of exons encoding 5'-untranslated sequences accounts for some of the heterogeneity in hPTHrP gene expression; however, the pattern and potential significance of alternative exon splicing at the 3'-end of the hPTHrP gene have not been systematically examined. We have used exon-specific primers and reverse transciptase-polymerase chain reaction to analyze hPTHrP gene expression in nonneoplastic human tissues and human tumors. PTHrP mRNA transcripts encoding PTHrP-(1-139), -(1-173), or -(1-141) were identified in the majority of tissues analyzed. PTHrP mRNAs containing either exon 6 [PTHrP-(1-139)] or exon 8 [PTHrP-(1-141)] sequences were considerably more abundant than mRNAs encoding PTHrP-(1-173) (exon 7) in nonneoplastic tissues. In contrast, a switch in the profile of the different hPTHrP mRNAs was detected in some neoplastic tissues, with an increase in mRNA transcripts encoding PTHrP-(1-173) and a decrease in mRNA transcripts encoding PTHrP-(1-141) observed in several tumors. The differential properties of specific PTHrP 3'-untranslated sequences encoded by unique exons were examined by creating a series of luciferase-PTHrP fusion genes and examining the contribution of PTHrP sequences to relative luciferase activity after transfection of heterologous cell lines. LUC-PTHrP fusion genes containing exon 6 or exon 8 sequences generated consistently higher luciferase activity than fusion genes containing sequences from exon 7. Furthermore, distinct differences in the relative profiles of luciferase activities were observed after transfection of several human, but not rodent, cell lines. These studies suggest that exon splicing at the 3'-end of the hPTHrP gene may be an important determinant of the biological complexity of hPTHrP gene expression.

Expression of annexin VI in A431 carcinoma cells suppresses proliferation: A possible role for annexin VI in cell growth regulation

Human A431 cells exhibit many characteristics typical of transformed cells, such as lack of contact inhibition and reduced growth factor requirement. We have used these cells as a model for the study of annexin VI function, since they do not normally express this protein. In this study we isolated two stably transfected clones, both of which were found to express annexin VI at physiological levels, and examined various growth parameters associated with the transformed phenotype. In low serum, normal A431 cells had doubling times similar to those observed in high serum. However, although the annexin VI transfectants grew only slightly more slowly than controls in high serum, their doubling time was significantly increased in low serum. Moreover, in low serum the annexin VI transfectants stopped proliferating after reaching confluence, indicating contact inhibition. Fluorescence activated cell sorting analysis revealed that the annexin VI + cells were growth arrested in the G 1 phase of the cell cycle when cultured in low serum, whereas annexin VI − clones exhibited the same proportion of mitotic cells in both low and high serum. Thus, expression of annexin VI in a heterologous cell line has a moderating influence on cell proliferation suggesting a possible role for annexin VI in cell growth regulation.

The chinook salmon gonadotropin II beta subunit gene contains a strong minimal promoter with a proximal negative element.

The salmon pituitary expresses two distinct gonadotropins, gonadotropin I (GTHI) and gonadotropin II (GTHII). These two hormones are synthesized in distinct pituitary cells and secreted at different stages during the reproductive cycle. To study the transcriptional regulation of the hormone-specific beta-subunit of GTHII (sGTHII beta) gene, approximately 3.5 kilobases of the 5'-flanking region was characterized and sequenced. The pituitary specificity of sGTHII beta was examined by analyzing sGTHII beta promoter activity in homologous primary pituitary cells derived from spawning rainbow trout and in a collection of heterologous cell lines. Various lengths of the 5'-flanking region of the sGTHII beta gene were ligated into a vector encoding the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting sGTHII beta/CAT chimeric constructs were analyzed using transient expression systems. Several constructs (-3500CAT, -1260CAT, -563CAT, and -39CAT) displayed readily detectable CAT activity in the pituitary cells derived from spawning male and female rainbow trout. In contrast, three of the constructs (-3500CAT, -1260CAT, and -563CAT) were expressed only at background levels in a variety of heterologous cell lines, suggesting that the 5'-flanking sequence of sGTHII beta contains information dictating its pituitary specificity. A silencer sequence (-95 to -35, pSil) was identified, which might function to repress sGTHII beta gene expression in the nongonadotropes or in the gonadotropes at developmental stages that precede final maturation and spawning.

Expression of Three Alternative Acetylcholinesterase Messenger RNAs in Human Tumor Cell Lines of Different Tissue Origins

To study the molecular mechanisms underlying the intensive expression of acetylcholinesterase (AChE) in different tumor types, we characterized levels and composition of its messenger RNA (mRNA) sequences in heterologous tumor cell lines, primary tumor biopsies, and normal fetal and adult tissues and determined their exon-intron origin within the corresponding ACHE gene. Reverse transcription followed by polymerase chain reaction (RT-PCR) revealed three alternatively spliced ACHE mRNAs in NT2/D1 teratocarcinoma, NCI-N-592 small cell lung carcinoma, TE671 medulloblastoma, K-562 erythroleukemia, and 293 transformed embryonal kidney cells. The three ACHE mRNAs include the principal species expressed in brain and muscle and two additional transcripts containing insertions of 751 or 829 residues downstream from the exon 4 domain. The inserted region, which represents an intron in brain and muscle, is expressed in the tumor cell lines either as a "readthrough" form or with 78 residues deleted from its 5′ end. A major band of 2.5 kb was labeled with ACHE cDNA in poly(A)+ RNA blots from medulloblastoma cells or brain tissue, whereas a PCR-amplified probe from the inserted domain labeled a 3.4-kb band but not the 2.5-kb band in poly(A)+ RNA from small cell lung carcinoma. The ACHE mRNAs including the alternative insertions were found only in cell lines with levels of the principal ACHE mRNA species equal to or higher than those in brain (1-10 molecules/cell), determined by following the kinetics of mRNA PCR amplification. Genomic DNA sequencing revealed that the inserted domains in the ACHE mRNAs expressed in the tumor cell lines encode C-terminal peptides of 40 and 14 residues. These include a free cysteine, terminate with the consensus HG element, and continue by a 29-residue-long C-terminal hydrophobic cleavable peptide, properties characteristic of precursors to phosphoinositide (PI)-linked proteins. In extention of the reported expression of PI-linked AChE in hemopoietic cells including K-562, our findings demonstrate the existence of ACHE mRNAs with the potential to encode one hydrophilic and two PI-linked forms of AChE in tumor cells from both hemopoietic and nonhemopoietic origins.

Transcriptional regulation of the Papilio polyxenes CYP6B1 gene.

Detoxification of host plant defensive compounds by larval Lepidoptera is mediated by cytochrome P450 monooxygenases (P450s) such as CYP6B1, which is expressed in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin. Baculovirus-mediated expression of two cloned CYP6B1 cDNAs in lepidopteran cell lines has demonstrated that CYP6B1 isozymes primarily metabolize the linear furanocoumarins, xanthotoxin and bergapten, and not angular furanocoumarins. To characterize the regulatory features of the CYP6B1 transcription unit, we have isolated the first full-length CYP6B1v3 genomic DNA clone from P. polyxenes. The open reading frame of this gene is interrupted by a single intron and is virtually identical to the previously characterized CYP6B1 cDNAs. Primer extension and ribonuclease protection analyses have localized the transcription initiation site to a point 28 nucleotides upstream from the AUG initiation codon. RNase protection analyses on RNA from larvae induced by linear and angular furanocoumarins indicate that transcription of the CYP6B1 gene is induced in insects significantly in response to xanthotoxin and only slightly in response to bergapten. Angular furanocoumarins, such as angelicin, which are not appreciably metabolized by the CYP6B1 gene product, do not significantly induce transcription of this gene. We conclude that this P450 gene is transcriptionally regulated in vivo by at least one of the substrates which the encoded protein metabolizes. Transient expression of CAT fusion constructs in transfected Sf9 lepidopteran cells demonstrates that nucleotides -1 to -838 upstream from the CYP6B1v3 transcription initiation site retain basal and xanthotoxin-inducible transcriptional activities in this heterologous cell line. These data clearly indicate that P. polyxenes has adapted to the presence of furanocoumarins in its host plants by evolving P450 isozymes and regulatory cascades which respond to specific toxins.

Generation of truncated brain AE3 isoforms by alternate mRNA processing.

AE3 gene is a member of the AE anion exchanger gene family that is expressed primarily in brain and heart. The principal product of the AE3 gene in rodent brain, FL-AE3p, when expressed in heterologous cell lines, gives rise to chloride-dependent changes in intracellular pH consistent with its operation as an anion exchanger. We have identified two novel isoforms of mouse AE3 that are generated by tissue-specific alternate RNA processing. One of these isoforms encodes a polypeptide, 14-AE3p, that corresponds to a portion of the NH2-terminal cytoplasmic domain of AE3. 14-AE3p lacks the entire transmembrane domain that-in FL-AE3p forms the anion exchange channel. Immunoblots with antibodies to the NH2- and COOH-termini confirm that FL-AE3 and 14-AE3 are expressed in rat brain as 160 kDa and 74 kDa polypeptides, respectively. Unlike FL-AE3p, however, 14-AE3p is insoluble in non-ionic detergent, suggesting a possible association with the cytoskeleton.

Identification of a novel zinc finger protein binding a conserved element critical for Pit-1-dependent growth hormone gene expression.

The growth hormone (GH) and prolactin genes require the pituitary-specific POU domain transcription factor Pit-1 for their activation. However, additional factors are necessary for the effective expression of these genes. Analysis of evolutionarily conserved sequences in the proximal GH promoter suggests the critical importance of one highly conserved element located between the two Pit-1 response elements. Mutation of this site decreases expression of a transgene in mice > 100-fold. We have identified a major activity binding to this site as a novel member of the Cys/His zinc finger superfamily, referred to as Zn-15. The Zn-15 DNA-binding domain comprises three zinc fingers separated by unusually long linker sequences that would be expected to interrupt specific DNA site recognition. Zn-15 synergizes with Pit-1 to activate the GH promoter in heterologous cell lines in which this promoter is only minimally responsive to Pit-1 alone. Our data suggest that functional interactions between the tissue-specific POU domain factor Pit-1 and this novel zinc finger factor binding to an evolutionarily conserved region in the GH promoter may constitute an important component of the combinatorial code that underlies the effective expression of the GH gene.

Distinct subdomains of human endothelin receptors determine their selectivity to endothelinA-selective antagonist and endothelinB-selective agonists.

The endothelin (ET) family of peptides acts via two subtypes of G-protein-coupled heptahelical receptors termed ETA and ETB, which have distinct rank orders of affinity to endothelin receptor agonists and antagonists. To delineate which portions of the receptor molecules determine ligand selectivity, we have constructed a series of chimeras between human ETA and ETB receptors and characterized the chimeric receptors expressed in heterologous cell lines by competitive radioligand binding analysis and by measuring agonist-induced transients of intracellular Ca2+. We demonstrate that the binding determinant for the ETB-selective agonists ET-3, BQ3020, and IRL1620 residues within the region spanning the putative transmembrane helices IV-VI and the adjacent loop regions. In contrast, the transmembrane helices I, II, III, and VII plus the intervening loop regions specify the selectivity for BQ123, an ETA-selective antagonist. BQ123 exhibited no detectable agonistic activity in all wild-type and chimeric receptors tested. A chimeric receptor that has the transmembrane helices IV-VI (and adjacent loops) from the ETB receptor inserted into the remaining regions from the ETA receptor binds both the ETA- and ETB-selective ligands with high affinities. Moreover, BQ123 competitively inhibits the binding of the amino-terminally truncated ETB agonists, 125I-BQ3020 and 125I-IRL1620, to this chimeric receptor, suggesting that BQ123 is a mimic of the carboxyl-terminal linear portion of endothelins. These findings indicate that there are at least two separable ligand interaction subdomains within the endothelin receptors.

Cell-specific posttranslational processing of the surfactant-associated protein SP-B

Pulmonary surfactant-associated protein B (SP-B) is a 9-kDa lung-specific protein expressed in alveolar epithelial type II cells and Clara cells. The protein markedly increases the surface activity of phospholipids and is an active component in some surfactants in clinical use. SP-B is produced from a 43-kDa precursor protein by proteolytic cleavage of flanking regions from both the NH2- and COOH-terminal ends of the active protein. In this study we have compared the nature of the posttranslational processing of the SP-B precursor in type II cells and in a heterologous cell line transfected with the SP-B precursor. We found that isolated type II cells produce the 9-kDa form of SP-B from the precursor through a series of intermediates detectable in the cell lysates. In contrast Chinese hamster ovary cells stably transfected with the full-length human SP-B precursor produce the precursor and a 26-kDa intermediate but not the 9-kDa protein. The precursor protein in both cell types is glycosylated with NH2-linked sugars. Our results suggest there is cell specificity in the posttranslational processing of the SP-B precursor.

Expression of a constitutively active erythropoietin receptor in primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burst-forming unit, and granulocyte/macrophage progenitor growth.

We tested the ability of a constitutively activated erythropoietin receptor [EpoR(R129C)] to alter the growth requirements of primary hematopoietic precursors that terminally differentiate in culture. Two recombinant retroviruses expressing EpoR(R129C), spleen focus-forming virus (SFFVc-EpoR) and myeloproliferative sarcoma virus (MPSVcEpoR), were used to infect fetal liver cells that served as a source of hematopoietic progenitors. Methylcellulose cultures were incubated in the absence of any added growth factors or in combination with selected growth factors. EpoR(R129C) completely abrogated the Epo requirement of erythroid colony-forming units to form erythrocytes after 2-5 days in culture and did not interfere with the differentiation program of these cells. In the absence of added growth factors EpoR(R129C) did not enhance erythroid burst-forming unit development. In contrast to experiments in heterologous cell lines, EpoR(R129C) did not render progenitor cells independent of interleukin 3 or granulocyte/macrophage colony-stimulating factor (GM-CSF). However, when progenitors were cultured with added steel factor, but not with interleukin 3 or GM-CSF, EpoR(R129C) augmented the growth and differentiation of erythroid bursts, mixed erythroid/myeloid, and granulocyte/macrophage (GM) colonies. Furthermore, both viruses were capable of expressing EpoR(R129C) in erythroid, mixed erythroid/myeloid, and GM colonies. Thus an aberrantly expressed and constitutively activated EpoR can stimulate proliferation of some GM progenitors. The ability of EpoR(R129C) to abrogate the Epo requirement of primary hematopoietic cells, but not the requirement for other cytokines, is consistent with the induction of erythroblastosis in vivo.

Tissue-specific and substrate-specific endoproteolytic cleavage of monkey pro-opiomelanocortin in heterologous endocrine cells: processing at Lys-Lys dibasic pairs.

This study compares the processing of pro-opiomelanocortin (POMC) at two Lys-Lys cleavage sites, located in the carboxy-terminal domain of the precursor, one site marking the amino terminus of beta-melanocyte-stimulating hormone (beta-MSH) and the other in the carboxy-terminus of beta-endorphin (beta E). These comparisons were carried out by transfecting monkey POMC cDNA into two heterologous cell lines: AtT-20, which endogenously expresses mouse POMC, and Rin m5F, which has been previously used as a host for transfected POMC. These cells lines are known to process POMC differently at Lys-Arg residues, though less is known about their Lys-Lys cleavage. Our results have demonstrated both tissue-specific and site-specific factors controlling Lys-Lys cleavage. The AtT-20 line appears not to perform either Lys-Lys cleavage. Rin m5F cells, on the other hand, fail to process the site at the carboxy terminus of beta E (beta E28-29) but do process, to a significant extent, the N-terminal site to beta-MSH. That this differential processing is unlikely to be due to a POMC conformation which would make the beta E site inaccessible was demonstrated by mutating the sites from Lys-Lys to Lys-Arg. With such mutants, Rin m5F cells fully processed at both locations. Interestingly, the mutant Lys-Arg sites were not fully processed by AtT-20 cells. These results are discussed in terms of the complement of processing enzymes expressed in each of the cell lines, as well as the role of residues surrounding the diabasic cleavage sites in determining the likelihood of proteolysis.

<b>CHARACTERIZATION OF PREPROTACHYKININ B PROCESSING IN HETEROLOGOUS ENDOCRINE CELL </b><b>LINES </b>

We have investigated endoproteolytic processing of protachykinin B and regulated release of neurokinin B following transfection into heterologous endocrine cell lines, AtT-20 and MIN6. Both of the transfected cell lines produced mature neurokinin B and secreted it in response to a secretagogue, 8-Br-cAMP. Northern blot analysis of PC3 and PC2, two recently cloned Kex2-like processing endoprotease, showed that AtT-20 cells expressed PC3 mRNA, while MIN6 cells expressed both PC2 and PC3 mRNAs. These data suggest that protachykinin B is processed to neurokinin B by at least PC3 within the regulated secretory pathway

Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.