In vivo effects of antisera to prolactin receptors in female rats.

Antisera generated in guinea pigs against partially purified PRL receptors derived from rabbit mammary glands were tested for their effect on PRL binding and the biological effects of PRL in vitro and in vivo. Several dilutions of either normal guinea pig serum or anti-PRL receptor serum were incubated in vitro with homogenates of rat ovaries. Although specific binding of PRL was inhibited as much as 90% by the anti-serum, normal guinea pig serum had little effect. There was no inhibition of binding of LH and FSH to ovaries by these antisera. When these antisera were administered to normal cycling rats, the most pronounced effect was an increase in the number of corpora lutea, presumably due to the prevention of the luteolytic effect of PRL. When one of these antisera was given to adult rats immediately after parturition, weight gain of their pups decreased significantly, possibly reflecting a decrease of milk yield. Thus, passive immunization of animals with anti-PRL receptor sera modifies the actions of PRL on some of its target tissues.     

Maintenance of prolactin (PRL) binding sites in rat liver cells in suspension culture: effect of PRL and of inhibitors of various cellular functions

An in vitro method to study the regulation of PRL receptors has been established using adult rat liver cells cultured in a continuous suspension in L-15 medium. PRL binding averaged 28.2 +/- 1.8% of the added labeled hormone per 10(6) cells in freshly isolated liver cells prepared from female rats treated with 17 beta-estradiol. When these cells were incubated at 37 C, binding rapidly declined by 50% at 10 h and 90% at 48 h. This rapid decline could be counteracted by the inclusion of ovine PRL (50 nM), which maintained initial PRL receptor levels up to 48 h of culture. Higher concentrations of PRL (2.5 microM) induced a rapid down-regulation, apparent at 2 and 10 h of culture. Cycloheximide (50 micrograms/ml) induced a slight diminution of control PRL receptor levels and partially reversed the effect of 50 nM PRL. Approximately 60% of the PRL receptors were resistant to the effect of cycloheximide. On the other hand, actinomycin D (10 micrograms/ml) had no effect on PRL receptor levels in control and only a very slight effect in PRL-treated cells. Dinitrophenol, which blocks metabolic oxidation, also partially reversed the effect of 50 nM PRL although it was without any significant effect on control levels. Chloroquine (100 microM) and colchicine (1 microM) failed to alter PRL binding either in the absence or presence of 50 nM PRL. Our results suggest that the existence of regulatory factors occurring in vivo, which are absent in the culture medium, could be responsible for the decline in PRL receptor levels in the control hepatocytes. PRL itself could be one of these factors. On the other hand, and in agreement with the putative actions of the drugs utilized, the mechanism of the PRL-induced maintenance of receptor levels appears to lie in part with an effect on receptor synthesis at the translational (ribsomal) level but to be independent of the internalization or of lysosomal degradation.     

Intragranular prolactin phosphorylation and kallikrein cleavage are regulated by zinc and other divalent cations

Rat prolactin (PRL) secretory granules contain enzymes for proteolytic cleavage and serial phosphorylation, but hormone cleavage products and phosphorylated PRL are not detected until just prior to exocytosis. Similarly, although PRL is stored in granules, in part, as high-mol-wt oligomers, PRL is primarily monomeric in the circulation. PRL secretory granules contain zinc, calcium, and magnesium, which inhibit depolymerization and dissolution of granules. Divalent cations also protect cysteine free thiol residues in the carboxy-terminal region of the intragranular hormone. The present studies examined the effect of removal and replacement of divalent cations on kallikrein cleavage and phosphorylation of secretory granule PRL. Kallikrein cleavage was assessed utilizing two experimental protocols. First, granules were treated with or without 3 mM EDTA, free hormone thiols were alkylated, the PRL was cleaved by kallikrein, and the small kallikrein-cleavage peptides were assessed by reversephase HPLC. No differences in hormone cleavage owing to removal of divalent cations were observed at this concentration of EDTA. Second, divalent cations in granules were reduced/removed by 10 mM EDTA/ 3 mM o-phenanthroline (OP), followed by addition of either 5 mM zinc, magnesium, calcium, or additional EDTA. Kallikrein cleavage was then initiated. In this instance, the extent of proteolysis was analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE) of the larger remnant PRL pieces. After treatment with 10 mM EDTA/3 mM OP, results indicated that cleavage between R174 and R175 (site 1) was unaffected by added cations or additional EDTA. Recovery of site 2 cleaved PRL (L1-K185) and site 3 cleaved PRL (L1-R188) was∼40% reduced by zinc, but unaffected by calcium or magnesium. Additional EDTA resulted in increased recovery of site 2 cleaved PRL, but no change in site 3 recovery, suggesting the presence of tightly bound intragranular zinc around site 2, even after the initial EDTA/OP treatment. Phosphorylation of PRL at S177 was studied using the same protocols. Phosphorylation was increased by added EDTA, even at 3 mM, and decreased by divalent cations, with no marked specificity for zinc observed. An additional experiment studied phosphorylation without exposure to kallikrein. Comparisons between the plus and minus kallikrein experiments showed kallikrein to have no apparent preference for unmodified or phosphorylated PRL. From the kallikrein cleavage and phosphorylation studies and modeling of PRL, we suggest D181 as a likely site for intragranular zinc coordination. When C189 and C197 are present as free thiols in intragranular PRL, these may also contribute to binding. Zinc coordination in this region of the molecule apparently regulates proteolytic processing by kallikrein, as well as contributing to the stability of the hormone storage forms.     

S179D prolactin primarily uses the extrinsic pathway and mitogen-activated protein kinase signaling to induce apoptosis in human endothelial cells

We have demonstrated that S179D prolactin (PRL) is potently antiangiogenic in vivo. Here, we examined apoptosis in human endothelial cells, using procaspase-8 and cytochrome c release as markers of the extrinsic and intrinsic pathways, respectively. Both pathways converge at caspase-3, which is responsible for cleavage of DNA fragmentation factor (DFF45). A 3-d incubation in 50 ng/ml S179D PRL quadrupled the number of early apoptotic cells; this effect was doubled at 100 ng/ml and became maximal at 500 ng/ml. DFF45 and procaspase 8 cleavage were detectable at 100 ng/ml. Cytochrome c, however, was unaffected until 500 ng/ml. The p21 increased at 24 h, whereas a change in p53 required both triple the time and higher doses. The p21 promoter activity was maximal at 50 ng/ml, whereas 500 ng/ml were required to see a significant change in the Bax promoter (a measure of p53 activity). Because S179D PRL and basic fibroblast growth factor (bFGF) have both been shown to activate ERK, the effect of S179D PRL on bFGF-induced ERK signaling was examined. S179D PRL blocked ERK phosphorylation in response to bFGF, whereas continued coincubation caused a delayed and prolonged activation of ERK. PD98059 inhibited this delayed activation of ERK and effects of S179D PRL on all measures except p53 levels or activity of the Bax promoter. We conclude that S179D PRL blocks bFGF-induced ERK signaling and yet uses ERK in a different time frame to elevate p21 and activate the extrinsic pathway. Prolonged incubations and high concentrations additionally activate the intrinsic pathway using an alternate intracellular signal.     

Evidence for a rapid stimulation of tyrosine kinase activity by prolactin in Nb2 rat lymphoma cells.

Studies were designed to determine if the activation of tyrosine kinases may be involved in the signal transduction pathway for PRL. Tyrosyl phosphorylation of cellular proteins was evaluated by western blot analysis of Nb2 cell proteins employing an antibody to phosphotyrosine. Physiological concentrations of ovine PRL (oPRL) had a pronounced effect on the tyrosyl phosphorylation of a 121 kDa protein. Increased tyrosyl phosphorylation of the 121 kDa protein was detectable with concentrations of oPRL as low as 0.5 ng/ml. Consistent with oPRL acting through a PRL receptor, hGH also stimulated tyrosyl phosphorylation of the 121 kDa protein when tested at concentrations between 5 and 20 ng/ml. In time course experiments, increased tyrosyl phosphorylation of the 121 kDa protein was apparent after a 5 min incubation with 20 ng/ml hGH, and maintained for at least one h. At higher concentrations of hGH (200 ng/ml), increased phosphorylation of the 121 kDa protein was clearly evident after only 1 min, indicating that tyrosyl phosphorylation of cellular proteins is an early event following ligand binding to the PRL receptor. Increased tyrosyl phosphorylation of proteins of 40, 90 and 55-65 kDa was also evident after incubation with hGH for 10, 10, and 60 min respectively. These findings are consistent with PRL-dependent tyrosine kinase activation being an early and perhaps initiating event in the signal transduction pathway for PRL in Nb2 cells.     

Biological activities and receptor interactions of des-Leu16 salmon and des-Phe16 human calcitonin

Analogs of salmon (des-Leu16 sCT) and human (des-Phe16 hCT) calcitonin were prepared in which the amino acid from position 16 was omitted. The biological activities were assessed in vivo in the rat hypocalcemic assay and in vitro by studying competition for binding of [125I]sCT and adenylate cyclase stimulation in human breast cancer cells (T 47D). Deletion from position 16 resulted in substantial loss of biological activity in each system, indicating the importance for a hydrophobic residue at position 16 in the intact calcitonin molecule.     

Genetic perturbation of the putative cytoplasmic membrane-proximal salt bridge aberrantly activates alpha(4) integrins

alpha(4) integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the alpha(IIb)beta(3) integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in alpha(4) integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine R(GFFKR) in alpha(4) integrins. In lymphocytes from knock-in mice (alpha(4)-R/A(GFFKR)), alpha(4)beta(1) and alpha(4)beta(7) integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of alpha(4)-R/A(GFFKR) lymphocytes. In vivo, alpha(4)-R/A(GFFKR) cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper alpha(4) integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration.     

1,25-Dihydroxyvitamin D3 receptors identified in the rat heart

Specific receptors for 1,25-dihydroxyvitamin D3, the active hormonal form of vitamin D3, were demonstrated in low salt chromatin preparations from normal rat hearts. Sucrose gradient analysis of KCl-extracted chromatin yielded a significant (P less than 0.005) peak of specific [3H]1,25-dihydroxyvitamin D3 binding in the 3.6S region. The peak of [3H]1,25-dihydroxyvitamin D3 binding was abolished by excess 1,25-dihydroxyvitamin D3, but not by 50 nM 25-hydroxyvitamin D3 nor by 1.0 microM levels of estradiol-17B, cortisol, or promegestone, demonstrating steroid specificity characteristic for such receptors. Upon Scatchard analysis this putative cardiac 1,25-dihydroxyvitamin D3 receptor yielded a single binding component with high affinity (KD = 0.36 nM) and low capacity (Nmax = 33 fmol/g tissue). Coupled with evidence for the presence of calcium binding proteins in this tissue, these observations suggest functional roles for 1,25-dihydroxyvitamin D3 and its receptors in cardiac muscle, possibly in regulating intracellular effects of calcium.     

The mechanism of action of cysteamine in depleting prolactin immunoreactivity

The thiol reagent cysteamine (CSH) depletes anterior pituitary cells of immunoreactive PRL both in vivo and in vitro. We examined the hypothesis that CSH affects either the solubility or immunoreactivity of PRL through a mechanism involving thiol-disulfide exchange. Adult female rats were treated with either CSH (300 mg/kg, sc) or an equimolar dose of ethanolamine as a control. Anterior pituitary glands were extracted in 0.1 M sodium borate buffer, pH 9.0. Treatment of pituitary extracts with beta-mercaptoethanol (BME) destroys the immunoreactivity of PRL. However, extraction in the presence of reduced glutathione or CSH of pituitaries of rats treated with CSH restores immunoreactive PRL to control levels. Extracts were also subjected to polyacrylamide gel electrophoresis (PAGE). On gels of pituitary extracts of CSH-treated rats, the band that comigrates with purified PRL is diminished compared to that in ethanolamine-treated controls. This is found regardless of whether the borate extracts are treated with BME. However, extraction of the pituitaries in sodium dodecyl sulfate-containing buffer followed by chemical reduction with BME restores the PRL band. Therefore, CSH acts on PRL through a thiol-related mechanism to yield a product that is poorly soluble in aqueous buffer at pH 9 and is poorly immunoreactive. Dispersed anterior pituitary cells in tissue culture were incubated with L-[35S]methionine to radiolabel newly synthesized peptides. These cultures were incubated in the presence of either CSH or ethanolamine. PAGE followed by autoradiography confirmed the above results obtained in vivo. Also, extracts of CSH-treated cultures were subjected to gel permeation chromatography. As determined by PAGE, at least some of the radiolabeled PRL can be recovered from void volume fractions by reduction with BME, indicating that CSH induces the formation, through disulfide exchange, of a high mol wt form of PRL, possibly PRL oligomers.     

Activation of JAK2 tyrosine kinase by prolactin receptors in Nb2 cells and mouse mammary gland explants.

One of the earliest cellular responses to prolactin (PRL) binding in Nb2 cells, a rat pre-T lymphoma cell line, is an increase in tyrosine phosphorylation of cellular proteins. In this work, immunologic techniques have been used to demonstrate that in Nb2 cells and in mouse mammary gland explants, JAK2, a non-receptor tyrosine kinase, is activated following stimulation with PRL. PRL stimulated tyrosine phosphorylation of JAK2 at times as early as 30 sec and concentrations of PRL as low as 0.5 ng/ml (2.5 pM) in Nb2 cells and 100 ng/ml (5 nM) in mammary gland explants. When JAK2 was immunoprecipitated from solubilized Nb2 cells or mammary gland explants and incubated with [gamma-32P]ATP, 32P was incorporated into a protein migrating with an apparent molecular weight appropriate for JAK2 only when cells had been incubated with PRL, indicating that JAK2 tyrosine kinase activity is exquisitely sensitive to PRL. In Nb2 cells, JAK2 was found to associate with PRL receptor irrespective of whether or not the cells had been incubated with PRL. These results provide strong evidence that JAK2 is constitutively associated with the PRL receptor and that it is activated and tyrosine phosphorylated upon PRL binding to the PRL receptor. These results are consistent with JAK2 serving as an early, perhaps initial, signaling molecule for PRL.     

Phenotypic expression of a novel C282Y/R226G compound heterozygous state in HFE hemochromatosis: Molecular dynamics and biochemical studies

Most adults affected with hereditary hemochromatosis are homozygous for a single point mutation of HFE (p.Cys282Tyr). Apart from the compound heterozygous state for the p.Cys282Tyr mutant and the widespread p.His63Asp variant allele, other rare HFE mutations can be found in trans and may have clinical impact. In the present report we describe the structural and functional consequences of a new mutation, namely the p.Arg226Gly which was inherited in trans with the p.Cys282Tyr allele in a patient affected with a mild iron overload. Because the R226G substitution is located in the vicinity of the normal Cys225S–S282Cys disulfide bond we initially investigated the structure of the variant by molecular dynamics techniques in order to estimate the effect of the mutation on the global structure of HFE domain α3. We found that the solvation free energy, hydrophobicity and formation of salt bridges are slightly modified with the global secondary structure of the α3 domain being conserved. In a previous paper, we demonstrated that the Q283P substitution leads to the loss of the normal Cys225S–S282Cys disulfide bridge. Similar to the Q283P substitution, the R226G substitution does not substitute a residue directly involved in the formation of the disulfide bridge. However, unlike the p.Gln283Pro variant which destroyed the normal disulfide bridge, the R226G mutation does not affect the normal Cys225S–S282Cys bridge. Furthermore based on cell line studies we clearly show that the mutation does not prevent cell surface localization, β2-microglobulin association and binding to transferrin receptor 1. This new compound heterozygous phenotype is very close to those of the C282Y/H63D compound heterozygous patients who display the biochemical hemochromatosis phenotype but with lower body iron stores than C282Y homozygotes. Our results do not exclude unknown genetic and/or metabolic factors that may act synergistically to increase the ferritin level.     

Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus

The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 A-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNA-binding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function.     

Ovarian dependence for pituitary tumorigenesis in D2 dopamine receptor-deficient mice

Hypophyseotropic dopamine exerts a tonic inhibitory tone on pituitary lactotrophs by the activation of dopamine D2 receptors (D2R). Ablation of D2R through gene knock-out approaches results in hyperprolactinemia and prolactinomas. This phenotype is more severe and develops more rapidly in female mice. We tested whether the female hypersensitivity is due solely to the loss of D2R inhibitory tone or concomitant stimulation by ovarian factors. C57BL/6J congenic D2R(-/-) mice were ovariectomized at 2 months of age and serum PRL levels were measured serially. Ovariectomy attenuated hyperprolactinemia and after 18 months, D2R(-/-) mice had average pituitary weights of 4 mg, compared with 60 mg in the intact group. 17beta-Estradiol did not restore PRL secretion or pituitary weight. Although the pharmacologic dose of estradiol slightly increased pituitary weight in wild-type and D2R(-/-) mice, it inhibited serum PRL in both intact and ovariectomized females and in castrated males. For comparison, we tested the estradiol response of wild-type 129S6/SvEv mice in the same paradigm and found the expected increase in pituitary weight and serum PRL. Our results demonstrate that the development of hyperprolactinemia and prolactinomas in mice lacking D2R is dependent on ovarian stimulation and likely involves a factor(s) in addition to estrogen. Furthermore, we showed that estradiol-induced proliferation and PRL secretion can be differentially regulated in a strain-specific manner. These findings illustrate the importance of genetic background when analyzing endocrine regulation in mutant mouse models.