microdissection-dissociation-murine-oviduct-individual-segment

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

University of California, Riverside

This manuscript discusses our studies to date concerning the effects of unmodified prolactin (PRL) and phosphorylated PRL on immune function. Most of the discussion refers to effects of changing the ratio of these two forms in maternal PRL on gamma delta T cell development in rat pups in utero, but limited experiments where adult animals have been directly treated are also discussed. The manuscript begins with some general background on gamma delta T cells and the different forms of PRL and then proceeds to a discussion of experimental findings. Results demonstrate that the ratio of unmodified to phosphorylated PRL during rat pregnancy is crucial to normal epidermal gamma delta T cell development in the pup thymus. Elevation of phosphorylated PRL in the dams, by administration of a recombinant molecular mimic of phosphorylated PRL, produces a defect in epidermalgamma delta T cell seeding and subsequent function in the offspring. In contrast, a functional defect is not seen for uterine gamma delta T cells in the offspring, a finding likely reflective of the continued availability of precursors to these cells after the fetal period. Preliminary results from treatment of the NZB/NZW mouse model of lupus with the two forms of PRL suggest opposing effects of unmodified and phosphorylated PRL on one measure of the disease.

Unmodified and phosphorylated prolactin and gamma delta T cell development and function.

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.

Prolactin receptor antagonists.

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.

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

We have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1-2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with β-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma. 

An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma.

A comprehensive understanding of prolactin's (PRL's) role in breast cancer is complicated by disparate roles for alternatively-spliced PRL receptors (PRLR) and crosstalk between PRL and estrogen signaling. Among PRLRs, the short form 1b (SF1b) inhibits PRL-stimulated cell proliferation. In addition to ligand-dependent PRLRs, constitutively-active varieties, missing the S2 region of the extracellular domain (ΔS2), naturally occur. Expression analysis of the ΔS2 version of SF1b (ΔS2SF1b) showed higher expression in histologically-normal contiguous tissue versus invasive ductal carcinoma. To determine the function of ΔS2SF1b, a T47D breast cancer line with inducible expression was produced. Induction of ΔS2SF1b blocked estrogen-stimulated cell proliferation. Unlike intact SF1b, induction of ΔS2SF1b had no effect on PRL-mediated activation of Stat5a. However induction inhibited estrogen's stimulatory effects on serine-118 phosphorylation of estrogen receptor α, serine-473 phosphorylation of Akt, serine-9 phosphorylation of GSK3β, and c-myc expression. In addition, induction of ΔS2SF1b increased expression of the cell cycle-inhibiting protein, p21. Thus, increased expression of ΔS2SF1b, such as we demonstrate occurs with the selective PRLR modulator, S179D PRL, would create a physiological state in which estrogen-stimulated proliferation was inhibited, but differentiative responses to PRL were maintained. 

Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma.

Controversy exists concerning the role of the long prolactin receptor (PRLR) in the progression of breast cancer. By targeting pre-mRNA splicing, we succeeded in knocking down only the long PRLR in vivo, leaving the short forms unaffected. Using two orthotopic and highly-metastatic models of breast cancer, one of which was syngeneic (mouse 4T1) to allow assessment of tumor-immune interactions and one of which was endocrinologically humanized (human BT-474) to activate human PRLRs, we examined the effect of long PRLR knockdown on disease progression. In both models, knockdown dramatically inhibited metastatic spread to the lungs and liver and resulted in increased central death in the primary tumor. In the syngeneic model, immune infiltrates in metastatic sites were changed from innate inflammatory cells to lymphocytes, with an increase in the incidence of tumor-specific cytotoxic T cells. Long PRLR knockdown in three-dimensional culture induced apoptosis of tumor-initiating/cancer stem cells (death of 95% of cells displaying stem cell markers in 15 days). We conclude that the long PRLR plays an important role in breast cancer metastasis. 

Anti-metastatic outcome of isoform-specific prolactin receptor targeting in breast cancer.

Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.

Prolactin inhibits a major tumor-suppressive function of wild type BRCA1.

Using multiple murine foster-nursing protocols, thereby eliminating placental transfer and allowing a distinction between dam- and pup-derived cells, we show that foster nursing by an immunized dam results in development of CD8(+) T cells in nonimmunized foster pups that are specific for Ags against which the foster dam was immunized (Mycobacterium tuberculosis or Candida albicans). We have dubbed this process "maternal educational immunity" to distinguish it from passive cellular immunity. Of the variety of maternal immune cells present in milk, only T cells were detected in pup tissues. Maternal T cells, a substantial percentage of which were CD4(+)MHC class II(+), accumulated in the pup thymus and spleen during the nursing period. Further analysis of maternal cells in the pup thymus showed that a proportion was positive for maternal immunogen-specific MHC class II tetramers. To determine the outcome of Ag presentation in the thymus, the maternal or foster pup origin of immunogen-responding CD8(+) cells in foster pup spleens was assessed. Whereas ∼10% were maternally derived in the first few weeks after weaning, all immunogen-responding CD8(+) T cells were pup derived by 12 wk of age. Pup-derived immunogen-responsive CD8(+) cells persisted until at least 1 y of age. Passive cellular immunity is well accepted and has been demonstrated in the human population. In this study, we show an arguably more important role for transferred immune cells: the direction of offspring T cell development. Harnessing maternal educational immunity through prepregnancy immunization programs has potential for improvement of infant immunity.

Maternal Milk T Cells Drive Development of Transgenerational Th1 Immunity in Offspring Thymus.

We have previously demonstrated lactational transfer of T cell-based immunity from dam to foster pup. In the short term, a significant part of transferred immunity is passive cellular immunity. However, as time progresses, this is replaced by what we have described as maternal educational immunity such that by young adulthood, all immune cells responding to a foster dam immunogen are the product of the foster pup's thymus. To reduce confounding factors, this original demonstration used congenic/syngeneic dam and foster pup pairs. In this study, we investigated lactational transfer of immunity to  in MHC class I-mismatched animals, as well as from Th1-biased dams to Th2-biased foster pups. Using immunized C57BL/6J dams, lactational transfer to nonimmunized BALB/cJ foster pups resulted in much greater immunity than direct immunization in 5-wk-old pups (ex vivo assay of pup splenocytes). At this age, 82% of immunogen-responding cells in the pup spleen were produced through maternal educational immunity. FVB/NJ nonimmunized foster recipients had a greater number of maternal cells in the spleen and thymus but a much larger percentage was Foxp3, resulting in equivalent immunity to direct immunization. Depletion of maternal Foxp3 cells from pup splenocytes illustrated a substantial role for lactationally transferred dam regulatory T cells in suppression of the ex vivo response in FVB/NJ, but not BALB/cJ, recipients. We conclude that lactational transfer of immunity can cross MHC class I barriers and that Th1 immunity can be imparted to Th2-biased offspring; in some instances, it can be greater than that achieved by direct immunization.

Article	Year
Unmodified and phosphorylated prolactin and gamma delta T cell development and function. Lupus\| PubMed ID: 11721700	2001
Prolactin receptor antagonists. Current opinion in investigational drugs (London, England : 2000)\| PubMed ID: 15898345	2005
Therapeutic potential of S179D prolactin--from prostate cancer to angioproliferative disorders: the first selective prolactin receptor modulator. Expert opinion on investigational drugs\| PubMed ID: 16989600	2006
An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma. Cancer letters\| PubMed ID: 24384092	2014
Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma. Cancer letters\| PubMed ID: 25527452	2015
Anti-metastatic outcome of isoform-specific prolactin receptor targeting in breast cancer. Cancer letters\| PubMed ID: 26095602	2015
Prolactin inhibits a major tumor-suppressive function of wild type BRCA1. Cancer letters\| PubMed ID: 26970274	2016
Maternal Milk T Cells Drive Development of Transgenerational Th1 Immunity in Offspring Thymus. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 27496970	2016
Lactation-Based Maternal Educational Immunity Crosses MHC Class I Barriers and Can Impart Th1 Immunity to Th2-Biased Recipients. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 28747348	2017

Ameae M. Walker

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