Biomedical subjects

Ameae M Walker

Publications and source records attributed to Ameae M Walker.

At least 19 recordsLinked to original sources

PubMed2006

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells.

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of beta-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of beta-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.

Adenoviridae↗

PubMed2006

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.

Annexin A5↗

PubMed2006

Unexpected effects of UVB in IL-10 transgenic mice: normalization of contact hypersensitivity response.

Solar radiation in the UVB range is absorbed primarily by the epidermal DNA where characteristic photodamage results in altered immune responses and mutagenic lesions. UVB exposure of the skin results in a profound upregulation of the anti-inflammatory cytokine, IL-10 and suppression of contact hypersensitivity (CHS). Given that IL-10 is produced after UVB exposure, and that antibodies against IL-10 have been shown to reverse UVB-induced immune suppression, we hypothesized that IL-10 transgenic mice would show an enhanced immune suppression in response to UVB. Using an IL-10 transgenic mouse model (IL-10tg), we examined the CHS response in unexposed animals and those exposed to UVB. Unexposed IL-10tg animals showed a diminished CHS response compared to wild-type. Surprisingly, however, when IL-10tg animals were exposed to UVB, the CHS response was not further suppressed, but rather was restored to the level observed in unexposed wild-type animals.

Animals↗

PubMed2006

Human chorionic gonadotropin beta (HCGbeta) down-regulates E-cadherin and promotes human prostate carcinoma cell migration and invasion.

BACKGROUND: Membrane-associated human chorionic gonadotropin beta (HCGbeta) is correlated with a poor prognosis in localized prostate adenocarcinoma. The relationship between HCGbeta and metastasis, however, is unclear. METHODS: To shed some light on the issue, two stable prostate carcinoma cell lines overexpressing HCGbeta, designated DU145 HCGbeta and PC3 HCGbeta, were created and compared with empty vector stably transfected DU145 and PC3 cells (control cells). RESULTS: HCGbeta expression resulted in a change in morphology; the cells were more elongated and had multiple pseudopodia, while the control cells were more rounded. This change in morphology was duplicated by incubating control cells in conditioned medium from the DU145 HCGbeta or PC3 HCGbeta cells, or by adding purified HCGbeta to control medium. The DU145 HCGbeta and PC3 HCGbeta cells were also less adherent than the controls, as assessed by the ease with which trypsin-EDTA could remove them from culture plates. Reduced adherence could be duplicated by incubation of control cells with either conditioned medium or purified HCGbeta. Western blot analysis showed that DU145 HCGbeta and PC3 HCGbeta cells expressed less E-cadherin than control cells and that a change of medium increased expression of E-cadherin. Addition of conditioned medium, or purified HCGbeta, to control cells down-regulated E-cadherin. Cell migration and invasion assays showed that DU145 HCGbeta and PC3 HCGbeta cells were more migratory and invasive than controls and that treatment of control cells with either conditioned medium or purified HCGbeta increased their migratory/invasive capacity. CONCLUSION: The data indicate that HCGbeta is directly responsible for changes in prostate carcinoma cells associated with an increased metastatic phenotype.

Cadherins↗

PubMed2006

Therapeutic potential of S179D prolactin--from prostate cancer to angioproliferative disorders: the first selective prolactin receptor modulator.

Increasing evidence suggests an important role for autocrine/paracrine prolactin in breast and prostate cancers and other disease states. Prolactin production in these extrapituitary sites is not governed by dopamine agonists, a finding that has spurred the production of prolactin receptor antagonists. This review focuses on one such antagonist, S179D prolactin, which was produced by mimicking a natural antagonist, phosphorylated prolactin. S179D prolactin is a very effective growth antagonist, partly because it inhibits signalling from unmodified prolactin and partly because it produces its own intracellular signal. This signal results in cell differentiation, cell-cycle arrest or apoptosis depending on dose, duration of treatment and cellular context. S179D prolactin is also a potent antiangiogenic and initial studies have shown it to be a potent anti-inflammatory agent. In light of these additional modes of action, it is suggested that S179D prolactin should now be more aptly referred to as a selective prolactin receptor modulator.

Animals↗

PubMed2006

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone.

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

Angiogenesis Inhibitors↗

PubMed2006

Different forms of prolactin have opposing effects on the expression of cell cycle regulatory proteins in differentiated mammary epithelial cells.

Prolactin (PRL) is a hormone that contributes to both the growth and differentiation of mammary epithelial cells, activities likely to impact breast cancer in opposite ways. Whether PRL causes growth or differentiation has been solely attributed to the coexisting steroidal environment, with PRL stimulating mammary gland growth during pregnancy, and then milk production after the postpartum drop in estrogen and progesterone. However, previous work from our laboratory has shown that the form of PRL may also be an important factor. During pregnancy, unmodified PRL (U-PRL) promotes mammary growth, while an increase in phosphorylated PRL, or administration of a molecular mimic of phosphorylated PRL (S179D PRL), inhibits growth. Unknown, however, is whether these forms of PRL have opposite effects on growth in the absence of steroids and whether effects are directly on mammary epithelial cells. To mimic the glandular epithelium in vitro, we used contact-inhibited, differentiated cells and showed that even with these minimally growing cells that treatment with U-PRL caused increased expression of cyclin D1 and cyclin-dependent kinase 4, increased activity of both cdk4 and cdk2, while having no effect on the inhibitory protein, p21. S179D PRL, by contrast, had no effect on cyclin D1 and cdk4 expression, but increased p21 expression and expression of the vitamin D receptor (VDR). We conclude that increased U-PRL or decreased phosphorylated PRL can directly affect cell cycle control proteins in relatively differentiated mammary epithelial cells, thereby implicating the balance between these two forms of PRL in the early promotion of breast cancer.

Animals↗

PubMed2005

Two wrongs can make a right: dimers of prolactin and growth hormone receptor antagonists behave as agonists.

Prolactin (PRL) and GH have two distinct binding sites (site 1 with high affinity; site 2 with low affinity) that each interact with a PRL receptor (PRLR) to form a functional receptor dimer that activates signal transduction. The G129R mutation in PRL and the G120R mutation in GH disrupt the structural integrity of site 2 such that the ligands retain the ability to bind to the first receptor with high affinity, but act as receptor antagonists. In this study, we examined the ability of monomeric and dimeric forms of these ligands, human (h) PRL and hGH, and their antagonists (hPRL-G129R and hGH-G120R) to 1) bind to PRLRs; 2) induce conformational changes in PRLRs; 3) activate signaling pathways associated with the PRLR; and 4) mediate cell proliferation in vitro. In contrast to monomeric hPRL-G129R, homodimeric hPRL-G129R induced PRLR dimerization; activated Janus family of tyrosine kinases 2/signal transducer and activator of transcription 5, Ras/Raf/MAPK kinase/Erk, and phosphatidylinositol 3-kinase/Akt signaling; and stimulated Nb2 cell proliferation. Similarly, homodimeric hGH-G120R was able to mediate signaling via the PRLR and to stimulate Nb2 cell proliferation. These experiments demonstrate that a ligand must have two functional binding sites, but that these may be site 1 plus site 2 or two site 1's, to elicit receptor-mediated signal transduction. The size of the ligand plays less of a role in receptor activation, suggesting that the extracellular portion of the PRLR (and possibly the GH receptor) is rather flexible and can accommodate larger ligands. These findings may have implications for designing multifunctional therapeutics that target this class of cytokine receptors.

Animals↗

PubMed2005

S179D prolactin increases vitamin D receptor and p21 through up-regulation of short 1b prolactin receptor in human prostate cancer cells.

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

Cell Cycle Proteins↗

PubMed2005

Transcriptional changes underlying the secretory activation phase of mammary gland development.

The secretory activation stage of mammary gland development occurs after parturition and converts inactive lobuloalveoli to active milk secretion. This process is triggered by progestin withdrawal and depends upon augmented prolactin (Prl) signaling. Little is known about the Prl-induced transcriptional changes that occur in the mammary gland to drive this process. To examine changes in the mammary transcriptome responsible for secretory activation, we have used transcript profiling of three mouse models that exhibit failure of secretory activation: knockout of galanin (a regulator of pituitary Prl production and a mammary cell autonomous modulator of Prl action); treatment with S179D Prl (a phosphoprolactin mimic); and knockout of a single Prl receptor allele. A significant reduction in expression was observed in genes belonging to 46 gene ontologies including those representing milk proteins, metabolism, lipid, cholesterol and fatty acid biosynthetic enzymes, immune response, and key transcription factors. A set of 35 genes, commonly regulated in all three models, was identified and their role in lactogenesis was validated by examining their expression in response to Prl stimulation or signal transducer and activator of transcription 5 knockdown in the HC11 mouse mammary cell culture model. The transcript profiles provided by these experiments identify 35 key genes (many for the first time) involved in the secretory activation phase of mammary gland development, show that S179D acts as an antagonist of Prl action, and provide insight into the partial penetrance of failed lactation in Prl receptor heterozygous females.

Alleles↗

PubMed2005

Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo- and hetero-pairs of long and short human PRL receptors in living human cells.

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP(2)) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP(2) when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb beta-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.

Amino Acid Substitution↗

PubMed2005

Prolactin receptor antagonists.

Efforts have been focused on the development of prolactin receptor (PRLR) antagonists, which, it is hoped, will be useful therapeutics for breast, prostate and possibly other cancers. Several approaches have been undertaken, including the production of pure antagonists and the combination of a PRLR antagonist with other antitumor molecules to increase efficacy. An alternative approach has been the development of a mimic of the natural PRL growth antagonist. This molecule is a combined antagonist/agonist since it inhibits signaling leading to growth, but promotes signaling leading to cell-cycle control, differentiation and apoptosis.

Humans↗

PubMed2004

Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids.

Soluble formazan assays are widely used for cell number assessment. However, in our hands, we observed frequent occasions in which the actual cell number was at odds with the assay reading. In this study, we have determined that (i) a large proportion of the reading obtained in commonly used culture media can be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls; (ii) the albumin present in 10% serum can reduce the assay absorbance by 50% so that an actual doubling of cell number can be obscured; and (iii) this latter effect is dependent on the concentration of fatty acids. To counter these problems, we have developed a protocol that gives consistent readings that are fully representative of cell number while retaining some of the original advantages of soluble formazan assays.

Animals↗

PubMed2003

Inhibition of prolactin (PRL)-induced proliferative signals in breast cancer cells by a molecular mimic of phosphorylated PRL, S179D-PRL.

Posttranslational modifications of prolactin (PRL), including phosphorylation, vary with physiologic state and alter biologic activity. In light of the growing evidence for a role for PRL in proliferation in mammary cancer, we examined the ability of a mimic of phosphorylated human PRL, S179D-PRL, to initiate signals to several pathways in mammary tumor cells alone and in combination with unmodified PRL. Unmodified PRL employed multiple pathways to increase cellular proliferation and cyclin D1 levels in PRL-deficient MCF-7 cells. S179D-PRL was a weak agonist compared with unmodified PRL with regard to cellular proliferation, cyclin D1 levels, and phosphorylation of signal transducer and activator of transcription 5 and ERKs. However, S179D-PRL was a potent antagonist of unmodified PRL to these endpoints. In contrast to the reduced levels of the long isoform of the PRL receptor observed in response to a 3-d incubation with unmodified PRL, S179D-PRL up-regulated expression of this isoform, 4-fold. These studies support the utility of this mutant as a PRL antagonist to proliferative signals in mammary epithelial cells, including a potential role in breast cancer therapeutics.

Animals↗

PubMed2003

Different biological effects of unmodified prolactin and a molecular mimic of phosphorylated prolactin involve different signaling pathways.

Previous work has shown that naturally phosphorylated prolactin antagonizes the growth-promoting activities of unmodified prolactin (U-PRL) and that this effect is duplicated by a molecular mimic, S179D PRL. At the same time, the S179D PRL is a superagonist with regard to expression of some PRL-regulated genes. We have asked whether the different activities of U-PRL and S179D PRL are the result of differential signaling. HC11 cells (a normal mouse mammary cell line) were grown to confluence, primed with hydrocortisone, and then exposed to the PRLs. A 15 min incubation of PRL-naive cells led to substantial tyrosine phosphorylation of Jak 2 and Stat 5a by U-PRL and an essentially equivalent Jak 2 activation by S179D PRL. The latter, however, was accompanied by reduced tyrosine phosphorylation of Stat 5a. EMSA analysis using a Stat 5 binding site showed both PRLs to cause equivalent binding of nuclear proteins and that most of what bound was complexed through Stat 5a. Phosphoamino acid analysis of Stat 5 showed S179D PRL to double the amount of serine phosphorylation versus that seen with U-PRL. Analysis of the MAP kinase pathway showed U-PRL capable of activation of ERKs 1 and 2 but that signaling via ERKs 1 and 2 was greater with S179D PRL. A 7-day incubation in either PRL increased beta-casein mRNA levels, but S179D PRL caused a 2-fold increase over that seen with U-PRL. The increase, over that seen with U-PRL, was blocked by the MAP kinase inhibitor, PD98059. After 7 days of treatment with S179D PRL, expression of the short PRL receptor was doubled, and signaling showed a greater dependence on the MAP kinase pathway (2.9-fold increase in ERK 1 and 2 activation). We conclude that although both PRLs use both pathways to some extent, U-PRL signals primarily through Jak 2-Stat 5 whereas S179D PRL signals primarily through the MAP kinase pathway especially after prolonged exposure. This is the first demonstration of differential involvement of signaling pathways by different forms of PRL.

Animals↗

PubMed2003

Opposite effects of unmodified prolactin and a molecular mimic of phosphorylated prolactin on morphology and the expression of prostate specific genes in the normal rat prostate.

BACKGROUND: In the current study, we have investigated the individual roles of unmodified, wild-type prolactin (WT PRL) and a molecular mimic of phosphorylated prolactin (S179D PRL) in the normal rat prostate. METHODS: In the first animal experiment, recombinant WT PRL and S179D PRL were delivered to adult male rats at a rate of 14 microg/kg per day for 3 weeks. In the second animal experiment, two subcutaneous (200 microg/kg) injections of long-acting forms of the two PRLs were given to adult male rats on day 1 and day 22 for a total of 5.5 weeks of treatment. RESULTS: The different forms of PRL had opposite effects on the normal rat prostate, independently of androgens. WT PRL promoted morphologic changes in prostate epithelium consistent with preparation for cell proliferation, whereas S179D PRL produced morphologic evidence of a more differentiated epithelium. Northern blot analysis of expression of the two major prostate specific proteins, prostatein and probasin, showed that WT PRL decreased, whereas S179D PRL increased, the expression of the mRNAs for these two proteins. At the same time, S179D PRL reduced both testosterone and dihydrotestosterone levels. CONCLUSION: We conclude that PRL is an important modulator of normal rat prostate biology and that different forms of PRL have specific functions. The molecular mimic of phosphorylated PRL, S179D PRL, is the most important in terms of epithelial cell differentiation.

Animals↗

PubMed2002

Pseudophosphorylated prolactin (S179D PRL) inhibits growth and promotes beta-casein gene expression in the rat mammary gland.

We have investigated the individual roles of unmodified prolactin (U-PRL) and a mimic of phosphorylated PRL (S179D PRL) in mammary development. Recombinant versions of the PRLs were delivered to rats throughout pregnancy at a rate of 6 microg/24 h per rat and to non-pregnant females at a rate of 24 microg/24 h per rat. Measurement of progesterone, corticosterone, and estradiol showed no effect of the administered PRLs on the levels of these other mammotropic hormones. Histological and morphometric analysis showed U-PRL to cause mammary growth, whereas S179D PRL inhibited growth. Molecular analysis demonstrated decreased beta-casein expression in the mammary glands of the U-PRL-treated animals at term and increased beta-casein expression in the mammary glands of the S179D PRL-treated animals. Superior beta-casein gene expression in response to S179D PRL versus U-PRL was confirmed in HC11 cells. We conclude that U-PRL is important for growth, whereas S179D PRL promotes at least one measure of differentiated function in the mammary gland.

Amino Acid Substitution↗

PubMed2002

p21-activated protein kinase gamma-PAK in pituitary secretory granules phosphorylates prolactin.

p21-activated protein kinase gamma-PAK phosphorylates prolactin (PRL) in rat pituitary secretory granules on Ser-177 and on the equivalent site, Ser-179, in recombinant human PRL. This is shown by comparison of phosphopeptide maps with the human PRL mutant S179D. gamma-PAK is present in rat and bovine granules as identified by in-gel phosphorylation of histone H4, and by immunoblotting. Thus, phosphorylation of PRL by gamma-PAK in granules produces the PRL molecule that has been shown to antagonize the growth-promoting activity of unmodified PRL, and is consistent with the identified role of gamma-PAK in the induction and maintenance of cytostasis.

Animals↗