Activin-A inhibits proopiomelanocortin messenger RNA accumulation and adrenocorticotropin secretion of AtT20 cells.

The complexity of corticotropic cell regulation by multiple central and peripheral factors is well recognized. The present study provides evidence for the participation of an additional factor in the regulation of this cell type of the anterior pituitary. Using the clonal AtT20 cell line as a model for corticotropes, homodimeric activin-A was observed to suppress basal ACTH secretion and POMC mRNA accumulation by approximately 50%. These effects required prolonged treatment with activin-A and were concentration dependent; the half-maximum concentration was in the range of 30-50 pM. Consistently, AtT20 cells were found to express specific high affinity binding sites for [125I]activin-A. The simultaneous addition of inhibin-A along with increasing concentrations of activin-A did not alter the characteristics of the inhibition of ACTH secretion by activin-A alone. This is in contrast to observations with gonadotropes of the anterior pituitary as well as a number of other cell types in which inhibin-A can partially antagonize the biological actions of activin-A. The results may suggest the participation of a subclass of activin receptors that mediate effects on ACTH secretion and POMC mRNA accumulation. As previously shown, the incubation of AtT20 cells with a synthetic glucocorticoid, dexamethasone, attenuated basal ACTH secretion and POMC expression in a concentration-dependent manner. The inhibition of both of these parameters by activin-A, however, was independent of glucocorticoids, because the two agents were additive in their actions. In addition to effects on secretion and mRNA levels, treatment with activin-A also inhibited the rate of proliferation of AtT20 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tubular early endosomal networks in AtT20 and other cells

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.     

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 1. [21-Desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin

The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [21-desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulin analogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-758]. The implications of these findings are discussed, and we conclude that the A20-A21 amide bond plays a significant role in the expression of the biological activity of insulin.     

Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.     

Gene expression in human cells with mutant insulin receptors

Insulin initiates its action by interacting with specific receptors on the plasma membrane of target cells. Mutations in these receptors cause the inherited insulin-resistant syndrome leprechaunism. Affected patients have severe intrauterine and post-natal growth restriction coupled with severe metabolic abnormalities. Fibroblasts from patients with leprechaunism have impaired in vitro growth, reflecting the growth restriction seen it in vivo. To determine the reason for the defective growth of cells from patients with mutant insulin receptors, gene expression was compared among fibroblasts from controls and patients with leprechaunism using DNA microarrays. Of the 12,626 human genes tested, cells from patients with leprechaunism had consistently increased mRNA for 151 genes and decreased mRNA for 51 genes. The level of expression of selected genes was independently confirmed by real time RT-PCR. Leprechaun cells had increased expression of several genes involved in metabolic functions, several of which were not previously known to be regulated by the insulin receptor. The absence of insulin receptors modified the expression of genes controlling apoptosis and cellular growth. Functional analysis indicated that cells from patients with leprechaunism had a normal response to apoptotic stimuli when mitochondrial potential and caspase activity were assayed. About 20% of the genes whose RNA was decreased in leprechaun cells coded for proteins involved in cell growth and differentiation. These results suggest that the insulin receptor is a physiologic regulator of several genes involved in intermediate metabolism even in human fibroblasts. Decreased expression of growth-promoting genes may explain the growth restriction of patients with severe insulin resistance.     

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.     

ACTH secretion by mouse corticotroph AtT20 cells is negatively modulated by the intracellular level of 7B2

7B2 is a pan-neuroendocrine protein known to facilitate the trafficking and activation of the prohormone proprotein convertase-2 (PC2). 7B2-null mice not only lack PC2 activity, but they also develop an adrenocorticotropic hormone (ACTH) hypersecretion syndrome, suggesting that 7B2 may regulate hormone secretion. To verify this possibility, we introduced into mouse corticotroph AtT20 cells a retroviral vector carrying either a sense or an antisense 7B2 transgene to induce higher and lower 7B2 expression, respectively. Relative to control AtT20 cells, 7B2-overexpressing cells released less ACTH following KCl-induced membrane depolarization, whereas cells expressing lower levels of 7B2 released relatively more, suggesting that 7B2-related peptides modulate regulated secretion in neuroendocrine cells.     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin

We have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A20-A21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A20-A21 amide bond can modulate biological activity independently of receptor binding affinity. The A20-A21 amide bond is thus considered as an important element in the "message region" of insulin.     

AtT20 cells express modified forms of pp60c-src

We have compared the properties of pp60c-src from the mouse pituitary tumor cell line, AtT20, and from mouse fibroblasts. In vitro, pp60c-src phosphotransferase activity from AtT20 cells is 2- to 3-fold that of mouse NIH 3T3 fibroblast pp60c-src. In analyzing the reason for this elevation in specific activity, we found that pp60c-src from AtT20 cells differs structurally in at least three ways from pp60c-src in fibroblasts. First, AtT20 cells and primary rat anterior pituitary cells express low levels of the neuronal form of pp60c-src. Second, pp60c-src from AtT20 cells is phosphorylated at two additional N-terminal serine residues. Last, AtT20 pp60c-src is phosphorylated to a lower overall stoichiometry.     

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.     

Basal adrenocorticotropin (ACTH) secretion is negatively modulated by protein phosphatase 5 in mouse pituitary corticotropin AtT20 cells

siRNA oligonucleotides for protein phosphatase 5 (PP5) were designed and transfected into mouse corticotroph AtT20 cells to induce lower PP5 expression levels. PP5-siRNA transfections (at 3 days) produced a approximately 50% down-regulation in targeted protein levels. PP5-underexpressing cells released significantly more ir-ACTH (10-12-fold) relative to baseline levels and promoted POMC release into the media. Neither CRF-mediated ACTH release nor dexamethasone-induced ACTH repression were affected in PP5-siRNA transfected cells. In summary, our observations suggest that endogenous PP5 can exert a negative modulatory effect on basal ACTH release in neurosecretion-competent AtT20 cells through a mechanism as yet unknown but which does not directly involve regulated CRF or glucocorticoid receptor-dependent pathways. However, PP5 may cause mis-sorting of POMC and POMC-derived peptides at the constitutive-like secretory pathway level in an unregulated manner. Such a missorting could lead to impaired processing of POMC.     

Endogenous hyperlactatemia and insulin secretion]

In the normal anesthetized dog, the endogenous hyperlactatemia induced either by intense muscular work or by a high dose of phenformin (20 mg/kg subtucaneously) is followed by an increase in the pancreaticoduodenal insulin output. A previous perfusion of sodium dichloroacetate (50 mg/kg. h) opposes the hyperlactatemia, and reduces or suppresses the increase in insulin output.     

Blocked coated pits in AtT20 cells result from endocytosis of budding retrovirions

AtT20 cells support the replication of two endogenous retroviruses, a murine leukemia virus and a mouse mammary tumor virus. On glass or plastic substrates, AtT20 cells grow in clumps. In this situation, retroviruses budding from the plasma membrane of one cell can, on rare occasions, be invested by coated pits in the plasma membranes of contiguous cells. These pits can invaginate to depths of 2,000-4,000 A within the cytoplasm drawing with them the viral buds which remain connected to their parental cells by tubular stalks, some of which are only 225 +/- 15 A in diameter. These stalks run down the straight necks of the pits from the buds to the parental cell surfaces. Several lines of evidence indicate that these unique structures are blocked such that neither endocytosis nor budding can go to completion, and that they persist for several hours. The properties of these blocked coated pits are relevant to models of both endocytosis and viral budding. First, they indicate that the invagination of a coated pit is not absolutely dependent on its pinching off to form a coated vesicle, but that uncoating appears to be dependent upon the generation of a free vesicle. Secondly, they suggest that the final stages in the maturation of a retroviral core into a mature nucleoid are dependent on the detachment of the bud from its parental cell and that the driving force of budding is the association of viral transmembrane proteins with viral core proteins. An explanation is offered to account for the formation of these structures despite the phenomenon of viral interference.     

Sta!le and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.     

Characterization of the TGN exit routes in AtT20 cells using pancreatic amylase and serum albumin

The AtT20 pituitary cell is the one that was originally used to define the pathways taken by secretory proteins in mammalian cells. It possesses two secretory pathways, the constitutive for immediate secretion and the regulated for accumulation and release under hormonal stimulation. It is in the regulated pathway, most precisely in the immature granule of the regulated pathway, that proteolytic maturation takes place. A pathway that stems from the regulated one, namely the constitutive-like pathway releases proteins present in immature granules that are not destined for accumulation in mature granules. In AtT20 cells proopiomelanocortin the endogenous precursor of the accumulated adrenocorticotropic hormone, is predominantly secreted in a constitutive manner without proteolytic maturation. In order to better understand by which secretory pathway intact proopiomelanocortin is secreted by a cell line possessing a regulated secretory pathway, it was transfected with rat serum albumin (a marker of constitutive secretory proteins), and pancreatic amylase (a marker of regulated proteins). COS cells were also transfected in order to serve as control of release by the constitutive pathway. It was observed that both the basal and stimulated secretions of albumin and proopiomelanocortin from AtT20 cells are identical. In addition, secretagogue stimulation when POMC is in transit in the trans-Golgi network decreases its constitutive secretion by 50%. It was also observed using cell fractionation and 20 degrees C secretion blocks that albumin and proopiomelanocortin are present in the regulated pathway, presumably in the immature granules, and are secreted by the constitutive-like secretory pathway. These observations show that stimulation can increase sorting into the regulated pathway, and confirm the importance of the constitutive-like secretory pathway in the model AtT20 cell line.     

Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.