Nb2 Lymphoma Cells Research Articles

Biological activity of phosphorylated and dephosphorylated bovine prolactin

Phosphorylation is a mechanism by which cells regulate structure and function of proteins. We have previously demonstrated in vivo synthesis and secretion of phosphorylated bovine prolactin (bPRL) from the pituitary, and have isolated and partially characterized the phosphorylated bPRL. In order to investigate the structure/function role of phosphorylation on the biological activity of bPRL, we compared the activities of nonphosphorylated and phosphorylated bPRL isolated from pituitaries, with bPRL provided by the NIDDK (NIDDK-bPRL) to stimulate Nb2 cell proliferation. Nonphosphorylated bPRL has activity similar to, although slightly lower than that of NIDDK bPRL (ED 50 = 7.03 pM and 22.8 pM, respectively). The activity of phosphorylated bPRL is significantly reduced (ED 50 = 1066 pM). Using Nb2 lymphoma cell homogenate, NIDDK and nonphosphorylated bPRLs are equally effective in competitive receptor binding assays ( K d = 0.252 and 0.269 nM, respectively). Phosphorylated bPRL does not compete for the PRL receptor at concentrations up to 65 nM. Following enzymatic removal of the phosphate group using alkaline phosphatese, there is an increase in the biological activity of phosphorylated bPRL (ED 50 = 73.3 pM) while the activity of nonphosphorylated BPRL remained unchanged following enzyme treatment (21.4 pM). We conclude that (1) structural changes induced by phosphorylation of bPRL are responsible for loss of bioactivity, (2) dephosphorylation of phosphorylated bPRL restores biological activity, and (3) the reduction in biological activity of phosphorylated bPRL is mediated by a decrease in receptor binding.

Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells.

Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.

Bioactivity and immunoreactivity of rat pituitary prolactin submitted to various extraction procedures.

Pituitary glands of rats, injected with estrogen to increase the prolactin (PRL) storage in secretory granules, were submitted to various extraction procedures for prolactin. Homogenization and centrifugation of pituitary tissue, in Tris-HCl buffer, pH 7.3, yielded a small amount of radioimmunoassayable prolactin, which increased remarkably after extraction in alkaline pH, disruption of granular membranes with Lubrol and specially after treatment with 2.5 mol/l urea. Nb2 lymphoma cell and pigeon crop sac bioassay (BA) revealed higher levels of bioactivity after extraction in Tris buffer, pH 7.3, in comparison to RIA, with BA/RIA ratios of 1.4 and 2.3, respectively. Bioassays of pituitary PRL extracted with Lubrol and alkaline medium, were less effective in quantifying PRL than RIA. Treatment of pituitary homogenates with urea produced the highest levels of PRL by both BA and RIA, with BA/RIA ratios close to 1. The thorough depolymerization of big PRL effected by urea produced the release of monomeric subunits which allows a complete quantification of the total content in the pituitary gland.

Nuclear translocation of prolactin: Collaboration of tyrosine kinase and protein kinase C activation in rat Nb2 node lymphoma cells

Recent evidence has suggested that prolactin (PRL), internalized by lactogen-dependent Nb2 lymphoma cells, is actively translocated to the nucleus where it binds to PRL receptors. Moreover, the mitogenic action of PRL in these cells has been separately linked to protein tyrosyl phosphorylation and activation of protein kinase C (PKC). Therefore, the coupling of PRL internalization and nuclear translocation to the activation of these signal transduction pathways was investigated. Results from control experiments indicated that 30% of internalized and 5% total cell-associated 125I-rat PRL could be recovered within nuclei obtained from Nb2 cells previously incubated with the radiolabel for 3 h at 37 degrees C. Furthermore, internalized PRL was found to be intact and not associated with any carrier proteins. Addition of tyrosine kinase (TK) antagonists, genistein or tyrphostin, significantly reduced cell surface binding, internalization, and nuclear translocation of 125I-rat PRL. In contrast, neither the level of cell-associated nor internalized hormone differed between cells treated with the PKC antagonists, staurosporine or calphostin C, and control cultures. Instead, PKC inhibition significantly reduced nuclear PRL translocation. The inhibitory effects of the TK and PKC antagonists on PRL internalization and nuclear translocation in intact Nb2 cells were verified by immunofluorescence microscopy in parallel experiments. In other experiments, each of the kinase inhibitors blocked PRL-stimulated Nb2 cell proliferation in a concentration-dependent manner. It is concluded that activated TK and PKC collaborate in the process of PRL internalization and translocation to the nucleus. TK activation may participate in PRL receptor binding or hormone internalization while activation of PKC appears to be required for its nuclear targeting. Since TK and PKC activation are required for lactogen-stimulated Nb2 cell proliferation, we suggest that a component of the mitogenic pathway in these cells is a direct nuclear interaction of PRL.

Prolactin (PRL)-dependent expression of a zinc finger protein-encoding gene, Gfi-1, in Nb2 lymphoma cells: constitutive expression in autonomous sublines

The proliferation of cultured rat Nb2 lymphoma cells is dependent on prolactin (PRL) acting as the principal growth factor. Previously, PRL-independent Nb2 sublines were obtained by PRL starvation of the parent line and cloning of surviving cells. Development of PRL independence was in some cases found to be associated with a reciprocal translocation involving chromosome 14 at breakpoint 14p22. In the present study, a novel, 14p22 zinc finger protein-encoding gene, Gfi-1, has been examined for a role in Nb2 cell proliferation. PRL-dependent Nb2 cells expressed the gene during active growth; in comparison, in stationary, early G1-arrested cells obtained by an 18 hr lactogen starvation, Gfi-1 gene expression was markedly decreased. Addition of PRL to such stationary cells led to induction of Gfi-1 gene expression within a few hr with a maximum in late G1. Actively growing cells from 5 different PRL-independent Nb2 sublines, cultured in chemically defined, mitogen-free medium, expressed the gene constitutively. In two sublines, carrying the 14p22 rearrangement, the gene was markedly overexpressed. The results suggest the Gfi-1 gene product has a regulatory role in Nb2 cell mitogenesis and that unscheduled activation could contribute to loss of PRL dependency.

Prolactin (PRL)-dependent expression of a zinc finger protein-encoding gene, Gfi-1, in Nb2 lymphoma cells: constitutive expression in autonomous sublines.

The proliferation of cultured rat Nb2 lymphoma cells is dependent on prolactin (PRL) acting as the principal growth factor. Previously, PRL-independent Nb2 sublines were obtained by PRL starvation of the parent line and cloning of surviving cells. Development of PRL independence was in some cases found to be associated with a reciprocal translocation involving chromosome 14 at breakpoint 14p22. In the present study, a novel, 14p22 zinc finger protein-encoding gene, Gfi-1, has been examined for a role in Nb2 cell proliferation. PRL-dependent Nb2 cells expressed the gene during active growth; in comparison, in stationary, early G1-arrested cells obtained by an 18 hr lactogen starvation, Gfi-1 gene expression was markedly decreased. Addition of PRL to such stationary cells led to induction of Gfi-1 gene expression within a few hr with a maximum in late G1. Actively growing cells from 5 different PRL-independent Nb2 sublines, cultured in chemically defined, mitogen-free medium, expressed the gene constitutively. In two sublines, carrying the 14p22 rearrangement, the gene was markedly overexpressed. The results suggest the Gfi-1 gene product has a regulatory role in Nb2 cell mitogenesis and that unscheduled activation could contribute to loss of PRL dependency.

Extracellular domain of prolactin receptor from bovine mammary gland: expression in Escherichia coli, purification and characterization of its interaction with lactogenic hormones

The cDNA of the extracellular domain of bovine prolactin receptor (bPRLR-ECD) was cloned and expressed at high yield as an insoluble protein in Escherichia coli. This protein was solubilized, refolded and purified to > 98% homogeneity yielding 80 mg of monomeric fraction per 2 litres of induced culture. Its molecular mass was 25.7 kDa, as determined by SDS-PAGE in the absence of reducing agent and 24 kDa by gel filtration on a Superdex column. Binding experiments revealed that bPRLR-ECD binds to human (h) GH (hGH) with high affinity, whereas its affinity for ovine (o) or bovine (b) prolactins (PRLs) was lower and for bovine placental lactogen (bPL) very low. The affinity of bPRLR-ECD for the latter three hormones was, however, much higher than that of membrane-embedded or solubilized bPRLR. The stoichiometries of interaction of bPRLR-ECD with hGH, oPRL, bPRL and bPL were determined by gel-filtration chromatography. Even at a 3:1 ECD excess, only 1:1 complexes were detected at microM concentrations of ECD and ligand. At an up to 32-fold dilution, the complexes with oPRL, bPRL, and particularly bPL, underwent progressive dissociation, whereas the complex with hGH remained stable. Although all four hormones exhibited nearly identical activity in the Nb2 lymphoma cell bioassay, the ability of bPRLR-ECD to inhibit hormonal mitogenic activities differed, generally reflecting its affinity for the respective hormones. In view of these and previous results, we suggest that, unlike in the GH:GHR-ECD interaction, neither the stoichiometry of interaction of bovine or other PRLR-ECDs nor the affinity constants can predict the biological potency of the different lactogenic hormones.

The Rabbit Mammary Gland Prolactin Receptor Is Tyrosine-phosphorylated in Response to Prolactin in Vivo and in Vitro

We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.

Effects of progesterone on prolactin, hypothalamic beta-endorphin, hypothalamic substance P, and midbrain serotonin in guinea pigs.

Unlike rats, but similar to primates, guinea pigs exhibit prolonged function of the corpus luteum and elevated progesterone secretion after ovulation. The gonadotropins, estrogen (E) and progesterone (P) have been examined throughout the guinea pig estrous cycle. However, neither prolactin secretion nor its regulation by steroid hormones has been characterized, perhaps due to the lack of a specific radioimmunoassay. beta-Endorphin (BE), substance P (SP), and serotonin (5-HT) increase prolactin secretion in rats and monkeys. BE and SP neurons in guinea pigs and 5-HT neurons in monkeys contain progestin receptors which could mediate neuroendocrine effects of steroid hormones. Therefore, the effects of E and P on prolactin, BE, SP, and 5-HT and its metabolite 5-HIAA were examined in guinea pigs which were ovariectomized, E treated (28 days), and E+P treated (14 days E+14 days E+P). The rat NB2 lymphoma cell line was used as a bioassay for serum prolactin. BE and SP levels were measured by radioimmunoassay in four hypothalamic areas: the preoptic region (POA), the mediobasal hypothalamus (MBH), the dorsomedial hypothalamus (DMH), and the mamillary bodies (MB). 5-HT and 5-HIAA were measured in the midbrain raphe area by high-pressure liquid chromatography. E alone had little effect on serum prolactin levels, but E+P significantly increased prolactin as compared with ovariectomized controls. The BE levels increased with E treatment and remained elevated with E+P treatment in MBH and POA. The BE content was stimulated in DMH and MB by E+P treatment and not with E alone. The SP content in MBH, DMH, and MB increased in E-treated guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin-Induced Phosphorylation and Nuclear Translocation of MAP Kinase in Nb2 Lymphoma Cells

The coupling of prolactin (PRL) receptor ligation to activation of mitogen-activated protein (MAP) kinase was sought in rat Nb2 lymphoma cells, a pre-T lymphocyte line dependent upon lactogens for proliferation. Addition of PRL (20 ng/ml) to Nb2 cells, growth arrested in the early G1 phase of cell cycle, stimulated rapid tyrosyl phosphorylation of MAP kinase (min). Phosphorylated MAP kinase subsequently translocated to the nucleus, with kinetics essentially identical to those demonstrated for nuclear accumulation of PRL. The rapidity of MAP kinase activation suggests an intermediary role for this enzyme in PRL receptor signalling. Moreover, nuclear translocation of MAP kinase provides an interactive mechanism by which PRL, together with its nuclear receptor, may regulate transcription requisite for mitogenesis.

Measurement of human urinary prolactin as a noninvasive study tool

We used a time-resolved solid-phase fluoroimmunoassay with a sensitivity of 25 ng/L on 40-fold-concentrated urines to measure urine prolactin (PRL) excretion. The nature of the immunoreactive material was verified to be PRL by: (a) column chromatography showing a monomeric 23-kDa peak; (b) similarity between fluoroimmunoassay and bioassay (Nb2 lymphoma cell) results; and (c) Western blot identification. In 20 normal subjects [serum PRL 6.8 (3.8-14.0) micrograms/L, median (and range)], urine PRL excretion was 0.15 (0.07-0.23) ng/h and 0.24 (0.15-0.54) micrograms/mol of creatinine. Urine values in seven hyperprolactinemic patients were all greater than the upper limit of normal. The correlation between urinary excretion rate and serum values was highly significant (r = 0.979; P < 0.001). These results indicate that a monomeric, immunologically reactive, biologically active form of PRL can be measured in urine at concentrations approximately 0.0005 that in serum. This urine PRL method may provide a practical tool for the repetitive, noninvasive study of PRL dynamics in field studies and in patients with reproductive disorders.

Regulation of heat-shock protein (hsp70) gene expression by hGH and IL2 in rat Nb2 lymphoma cells

A comparative study of hGH and IL2 post-signaling effects, as examined by RNA expression (Nb29) and protein levels of the heatshock protein hsp70, was performed in a hormone-dependent rat lymphoma cell line, Nb2-llC. Optimal doses of hGH or IL2 increased Nb29 expression in a dose-dependent manner. Addition of both mitogens to cell cultures affected Nb29 expression and mitogenesis synergystically, indicating a possible interaction between the post-receptoral mechanisms of these mitogens. Pretreatment of the cells with cholera toxin (CT) inhibited Nb29 expression, protein levels and mitogenesis of hGH- or IL2-induced cells up to 50%, indicating the involvement of Gs-proteins in the post-signaling processes of both hGH and IL2. Incubation of cell cultures with low concentrations of pertussis toxin (IAP) (0.01 ng/ml) markedly increased Nb29 expression in hGH but not in IL2-induced cells, suggesting specific involvement of the Gi-protein in post-signaling processes of hGH-induced cells. Addition of the PKC activator 12- O-tetra-decanoyl phorbol ester (TPA) to control cell cultures markedly increased the expression of Nb29 RNA levels but not mitogenesis, indicating that induction of these proteins in the cells is not sufficient for cell proliferation. Furthermore, incubation of hGH- or IL2-induced cells with the potent PKC inhibitor staurosporin (ST) decreased the levels of Nb29 in both hGH- and IL2-induced cells, although the effect of the mitogens differed significantly in their inhibition slopes. These results indicate that activation of PKC is one of the signaling pathways differentially involved in hGH and IL2 stimulation of cells. A model for post-receptoral processes is therefore proposed in which hGH and IL2 counteract in more than one signaling pathway, leading to the final stimulation and mitogenesis of Nb2 cells.

Prolactin bioactivity and the duration of lactational amenorrhea.

In our population, only half of fully nursing women remain amenorrheic 6 months postpartum. The other half recover their menstrual cycles between 90-180 days postpartum in spite of a high suckling frequency and elevated immunoreactive PRL (IR-PRL) concentrations. To further investigate the association of PRL with the recovery of ovarian function, we compared PRL bioactivity (BIO-PRL) 3-4 months postpartum in fully nursing amenorrheic women who subsequently experienced long (> 180 days; n = 5) or short (< 180 days; n = 5) lactational amenorrhea. In the present study, BIO-PRL in plasma was measured by the Nb2 lymphoma cell assay in samples taken before and 30 min after a suckling episode at 0800, 1600 and 2400 h. Women in the long amenorrhea group had higher overall mean BIO-PRL (mean +/- SE, 129.9 +/- 12.1 micrograms/L) than nursing women in the short amenorrhea group (66.6 +/- 5.2 micrograms/L; P < 0.05). Mean basal values were similar, but the women in the long amenorrhea group had more BIO-PRL in response to suckling (160.1 +/- 4.0 vs. 71.9 +/- 6.7 micrograms/L; P < 0.05). Compared with their respective basal values, nursing women in the long amenorrhea group demonstrated increased BIO-PRL in response to suckling, whereas the other group did not. The relationships between BIO-PRL and IR-PRL were similar in the two groups of nursing women before suckling. However, after suckling, the long amenorrhea group had significantly higher BIO-PRL levels than IR-PRL levels (P < 0.05, by likelihood test) than the short amenorrhea group. This suggests that suckling differentially changes in each group either the composition of PRL present or substances that may modify the bioactivity of PRL in plasma.

Serum lactogens possessed normal bioactivity in patients with lactation insufficiency

Insufficient breast milk is the most common reason for premature termination of breast-feeding. The causes of lactation insufficiency are usually multifactorial; in a small percentage of cases it is due to primary lactation failure of unknown origin. The aim of this study was to investigate whether lactation insufficiency of unknown origin could be caused by serum lactogens that had reduced biological activity. Women with lactation insufficiency of unknown origin and normal lactating controls were subjected to a standardized breast-feeding test for assessment of milk production. Thirty minutes later, serum samples were obtained for determination of total lactogen bioactivity, using an in-vitro bioassay, and levels of prolactin (PRL) and growth hormone (GH) using radioimmunoassay (RIA). Twelve lactating mothers with a clinical diagnosis of lactation insufficiency of unknown origin were compared with 12 matched mothers with normal lactation. The Nb2 lymphoma cell bioassay was used to measure total lactogen bioactivity in sera. Conventional RIA kits were used to estimate serum PRL and GH concentrations. Mean milk yield on standardized test feed was 21.6 ml for patients and 146.5 ml for controls. In both patient and control groups the total serum lactogen bioactivity ranged from about 150 to 5000 mIU/l, while the serum RIA (PRL+GH) levels ranged from about 350 to over 7000 mIU/l. There was no evidence of lactogens with reduced bioactivity in the patients' sera. Lactation insufficiency in the women studied cannot be explained by serum lactogens that possess unusually low bioactivity.

Prolactin activates the interferon-regulated p91 transcription factor and the Jak2 kinase by tyrosine phosphorylation.

The prolactin (PRL) receptor is a member of the family of cytokine receptors that lack intrinsic tyrosine kinase activity but contain two conserved cysteines in their N-terminal regions and a WSXWS motif adjacent to their transmembrane domains. In a manner similar to the interferons (IFNs), exposure of cells to PRL results in tyrosine phosphorylation of several cellular proteins and the rapid transcriptional induction of the IFN regulatory factor 1 gene. In this communication, we demonstrate that treatment of rat Nb2 lymphoma cells with PRL activates a latent protein factor so that it binds to an enhancer in the IFN regulatory factor 1 gene. This enhancer has been shown to be required for IFN-gamma-activated expression of this gene. PRL-induced assembly of the DNA binding complex, PRL-stimulated factor, required tyrosine phosphorylation. PRL-stimulated factor contained at least one protein that was antigenically similar to the p91 transcription factor, a component of several transcription complexes required for cytokine-activated gene expression. PRL not only induced the tyrosine phosphorylation of p91 but also induced tyrosine phosphorylation of Jak2, a tyrosine kinase required for IFN-gamma-activated gene expression. These results provide evidence for a signaling mechanism, some of whose components are shared by both PRL and IFN-gamma receptors, that results in the expression of early response genes.

Growth signaling and JAK2 association mediated by membrane-proximal cytoplasmic regions of prolactin receptors

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.

Vanadium activates or inhibits receptor and non-receptor protein tyrosine kinases in cell-free experiments, depending on its oxidation state. Possible role of endogenous vanadium in controlling cellular protein tyrosine kinase activity

We have shown that vanadium mimics several insulin effects in rat adipocytes, via a staurosporine sensitive cytosolic protein tyrosine kinase (CytPTK; Shisheva, A., and Shechter, Y. (1993) J. Biol. Chem. 268, 6463). Here we demonstrate that vanadium effects on protein tyrosine kinases are preserved after cell disintegration. Vanadium inhibits or activates protein tyrosine kinases depending on its oxidation state and the tyrosine kinase studied. Vanadyl (4+) but not vanadate (5+) inhibits receptor tyrosine kinases such as the insulin receptor (IC50 value = 23 +/- 4 microM) and the insulin-like growth factor-I receptor (IC50 = 19 +/- 3 microM). Inhibition is non-competitive with respect to ATP, Mn2+, or substrate concentrations. Preincubation of adipocytes with vanadyl (0.4 mM), and staurosporine (which arrests the cytosolic enzyme) substantially inhibited insulin-stimulated lipogenesis. Vanadyl is readily oxidized to vanadate by hydrogen peroxide. In contrast, CytPTKs were poorly inhibited by vanadyl, and vanadate stimulated several CytPTKs 2-6 fold. CytPTK derived from rat adipocytes, liver and brain were activated, and CytPTK from Nb2 lymphoma cells was not affected. CytPTK extracted from insulin-responsive tissues are more sensitive to vanadate activation (ED50 = 3 +/- 0.7 microM), whereas the brain enzyme is less sensitive (ED50 = 27 +/- 3 microM). Tungstate, molybdate, and phenylarsine oxide also stimulate CytPTK, suggesting that the vanadate effect is secondary to inhibiting protein phosphotyrosine phosphatases. This study supports a working hypothesis implicating the intracellular vanadyl pool in modulating CytPTK activity. Any physiological conditions converting vanadyl to vanadate (i.e. H2O2 production) will activate CytPTK and consequently CytPTK-dependent bioeffects.

Inhibition of growth hormone bioactivity by recombinant human growth hormone-binding protein in the eluted stain assay system.

The effects of a recombinant human GH-binding protein (rhGHBP; amino acids 1-238) on GH stimulation of rat Nb2 lymphoma cells were examined with an eluted stain assay system (ESTA). This precise bioassay utilizes the colorimetric reduction by stimulated Nb2 cells of a yellow tetrazolium salt (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan as its end-point. The use of a lactogenic bioassay allowed the investigation of hGHBP specificity for human GH (hGH) as opposed to prolactin. rhGHBP inhibited pituitary hGH bioactivity in a dose-dependent manner. No significant inhibition of prolactin or ACTH bioactivity occurred. It was confirmed that recombinant 20 kDa hGH also stimulated the Nb2 cells and that its relative potency was approximately 10% of that of pituitary-derived hGH. Stimulation by 20 kDa hGH was also inhibited by rhGHBP. The highly quantitative ESTA system demonstrated that the binding protein inhibited in a competitive manner. hGH activation of the Nb2 cells did not appear to be governed by a Michaelian first-order reaction. As might then be anticipated, the concentration of rhGHBP required for 50% inhibition of GH bioactivity (IC50) changed with agonist concentrations for both 20 kDa and 22 kDa hGH. However, with equimolar concentrations of these two isohormones, the IC50 of the binding protein was virtually identical. Potentiation of hGH bioactivity in vivo by low concentrations of hGHBP has been reported but was not observed in our in vitro system when tested over a wide range of binding protein concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression, purification, and characterization of recombinant rat placental lactogen-I: a comparison with the native hormone.

Rat placental lactogen-I (rPL-I), a member of the PRL/GH gene family, is produced by giant cells in the early trophoblast. The small amount of early placental tissue has limited the purification of rPL-I from this source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (CHO) cells and in these studies have compared the recombinant protein with the native rPL-I. Using an affinity column composed of monoclonal antibody to rPL-I coupled to Sepharose 4B, we have purified rPL-I from four sources: 1) recombinant rPL-I produced and secreted in rPL-I-transfected CHO cells, 2) nonglycosylated recombinant PL-I produced by adding tunicamycin (10 microM/ml medium) to rPL-I-transfected CHO cells, 3) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) serum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed nine subforms with increasing mol wt [approximately 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (approximately 23 kDa) were evident in the nonglycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (approximately 37 kDa) and more acidic pI. When analyzed by the Nb2 lymphoma cell bioassay, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipotent with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as active as ovine PRL in the Nb2 assay. A RIA was established for rPL-I. The variant rPL-Iv, displayed nonparallel displacement of [125I]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum levels of rPL-I by RIA after the injection of recombinant-rPL-I into adult female Sprague-Dawley rats revealed a half-life of 9 min for the recombinant protein compared to 7.8 min for the choriocarcinoma-derived hormone. In conclusion, we have shown that although CHO cells will glycosylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to those of the native, placenta cell-derived hormone.

Expression, purification, and characterization of recombinant rat placental lactogen-I: a comparison with the native hormone

Rat placental lactogen-I (rPL-I), a member of the PRL/GH gene family, is produced by giant cells in the early trophoblast. The small amount of early placental tissue has limited the purification of rPL-I from this source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (CHO) cells and in these studies have compared the recombinant protein with the native rPL-I. Using an affinity column composed of monoclonal antibody to rPL-I coupled to Sepharose 4B, we have purified rPL-I from four sources: 1) recombinant rPL-I produced and secreted in rPL-I-transfected CHO cells, 2) nonglycosylated recombinant PL-I produced by adding tunicamycin (10 microM/ml medium) to rPL-I-transfected CHO cells, 3) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) serum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed nine subforms with increasing mol wt [approximately 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (approximately 23 kDa) were evident in the nonglycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (approximately 37 kDa) and more acidic pI. When analyzed by the Nb2 lymphoma cell bioassay, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipotent with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as active as ovine PRL in the Nb2 assay. A RIA was established for rPL-I. The variant rPL-Iv, displayed nonparallel displacement of [125I]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum levels of rPL-I by RIA after the injection of recombinant-rPL-I into adult female Sprague-Dawley rats revealed a half-life of 9 min for the recombinant protein compared to 7.8 min for the choriocarcinoma-derived hormone. In conclusion, we have shown that although CHO cells will glycosylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to those of the native, placenta cell-derived hormone.