Integration of progesterone receptor mediated rapid signaling and nuclear actions in breast cancer cell models: role of mitogen-activated protein kinases and cell cycle regulators.

Progesterone receptor (PR) isoforms are dual functioning steroid hormone receptors, capable of activation of target gene transcription, and rapid stimulation of membrane-initiated intracellular signaling cascades. Herein we provided a retrospective of our recent work investigating the role of progestin-activated intracellular signaling pathways on cell cycle progression in breast cancer cell models. We show that progestin-induced S-phase entry and upregulation of selected target genes, including cyclin D1, are MAPK-dependent events. Further experiments conducted with mutant PRs defective in either the transcriptional response (PR-S294A) or activation of c-Src-dependent intracellular signaling to MAPKs (PR-mPro) confirmed that the proliferative response of breast cancer cells to progestins is largely dependent on the ability of PR to rapidly activate Erk 1/2 MAPKs. During progestin-stimulated cell cycle progression, elevated cdk2 levels and activity target multiple phosphorylation sites on PR. Phosphorylation of Ser400 augments PR nuclear localization and mediates increased PR transcriptional activity in the absence of hormone, while the cdk inhibitor, p27, reversed these effects. Together, our data illustrate the versatility of PR as regulatory signaling molecules that also act as sensors for multiple kinase pathways, and suggest that progestins influence changes in breast cancer cell gene expression and proliferation via integration of PR functions as both ligand-activated transcription factors and rapid initiators of intracellular signaling pathways.

Phosphorylation of progesterone receptor serine 400 mediates ligand-independent transcriptional activity in response to activation of cyclin-dependent protein kinase 2.

Human progesterone receptors (PR) are phosphorylated by cyclin-dependent protein kinase 2 (CDK2) at multiple sites, including Ser400. Herein, we have addressed the significance of phosphorylation of this residue. PR phospho-Ser400-specific antibodies revealed regulated phosphorylation of Ser400 in response to progestins and mitogens, and this correlated with increased CDK2 levels and activity. Expression of cyclin E elevated CDK2 activity and downregulated PR independently of ligand. Similarly, overexpression of activated mutant CDK2 increased PR transcriptional activity in the absence and presence of progestin. Mutation of PR Ser400 to alanine (S400A) blocked CDK2-induced PR activity in the absence, but not in the presence, of progestin. PR was unresponsive to activated CDK2 in breast cancer cells with elevated p27, and RNA interference knock-down of p27 partially restored CDK2-induced ligand-independent PR activation. Similarly, in p27(-/-) mouse embryonic fibroblasts, elevated CDK2 activity increased wild-type (wt) but not S400A PR transcriptional activity in the absence of progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules.

Robustness into advanced age of atopy-specific mechanisms in atopy-prone families.

We evaluated atopy-associated parameters in 1,099 people (aged 6-84 years) from families with history for atopy. All were tested for serum total immunoglobulin E (IgE) and allergen sensitivity by skin prick test. Specific IgE tests were done in randomly selected families. There was a decline with age in serum total IgE values, and relative atopy "incidence rates" were slightly lower among those older than 60 years. However, there was no change with age in sensitivity or severity of atopy. Among those sensitized to ragweed (Ambrosia artemisilfolia), there was no age-associated change in IgE levels specific to Amb a 1, a major allergen extracted from ragweed, and no change in the binding affinity of IgE for the Amb a 1 allergen. Among families with atopic histories, the underlying atopic mechanisms are particularly robust, and the atopic propensity remains into advanced age. In addition, established atopic responses may be focused in an immune system compartment either independent of or minimally influenced by T-cell activity.

Allergen skin test reaction patterns in children (

BACKGROUND: Genetic studies of atopy rely upon evidence of abnormal IgE production, usually elevated total IgE or skin prick test (SPT) reactions. However, these measures may change with subject age. METHODS: We screened 1,099 members of atopic families (aged 6-87 years) by serum total IgE and SPT for 14 allergens. For those SPT negative, we screened for Amb a 1- and Der p 1-specific IgE. Der p 1 IgE-Der p 1 allergen binding affinities were done on randomly selected subjects. RESULTS: There were significantly fewer atopics 10 years old (75.8%) based upon any SPT-positive result. Children 10 years old = 82.3%). Among those SPT-positive for house dust mite extract, there was a positive correlation between Der p 1 binding affinity and the wheal area of the house dust mite extract. There was a positive correlation between the number of SPT-positive reactions and total IgE for both age groups. However, there was only a significant relationship between SPT-positive wheal area and total IgE for those >10 years old and no apparent relationship between wheal area and total IgE for those

Cross-talk between growth factor and progesterone receptor signaling pathways: implications for breast cancer cell growth.

Breast cancers often have increased mitogen-activated protein kinase (MAPK) activity; this pathway influences breast cancer cell growth in part by targeting steroid hormone receptors. Activation of p42 and p44 MAPKs increases progesterone receptor (PR) transcriptional activity in the presence of progestins, and triggers their rapid down-regulation by the ubiquitin-proteasome pathway. In turn, progestins increase the expression of type I growth factor receptor tyrosine kinases that feed into MAPK activation. Most recently, progestins have been shown to activate the p42/p44 MAPK module in a progesterone receptor (PR) dependent manner, but independently of their function as transcription factors. Indeed, mechanisms of bi-directional cross-talk between these two pathways are becoming well-documented. In this reveiw we provide an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using examples from our work and others with human PR as a model receptor. We highlight the regulation of PR by phosphorylation and the role of intracellular protein kinases as key mediators of PR action. Cross-talk between growth factor and PR-mediated signaling events is an important means by which growth regulatory genes may be coordinately regulated, and may contribute to the growth and development of hormonally responsive normal breast tissue and to breast cancer progression.

Variable binding affinities for allergen suggest a 'selective competition' among immunoglobulins in atopic and non-atopic humans.

Atopy is a persistent, aberrant humoral response to certain classes of proteins (allergens) characterized by the presence of allergen-specific IgE. Yet, in both atopic and non-atopic individuals, allergen-specific responses involving the IgA and IgG subclasses have been observed, which evidence does not support models suggesting inherited differences in sensitivity to certain protein classes. Using the major ragweed component Amb a 1 as a model allergen, we assessed the humoral responses in three groups of unrelated donors: (A) atopic, ragweed sensitive; (B) atopic, but not ragweed sensitive; (C) non-atopic. As expected, Amb a 1-specific IgE was present in group A only. However, there were essentially no differences in the relative proportions of Amb a 1-specific IgA(1,2) and IgG(1-4) among the groups. We also determined the Amb a 1 binding affinities for IgG(1) and IgG(4) in the three groups, and compared these to Amb a 1-specific IgE binding affinities in group A. Group A donors' Amb a 1-IgE had extremely high affinities (10(8) to 10(11)M(-1)), but their Amb a 1-IgG(1) and Amb a 1-IgG(4) affinities were significantly lower (10(7) to 10(10)M(-1)). The average IgG(4) binding affinities in groups B and C were slightly higher than that of IgG(4) in group A, although not statistically significant. However, the IgG(1) affinity for Amb a 1 among group C, non-atopic donors was significantly elevated and comparable to the IgE affinity observed in group A, ragweed atopics. Inhibition studies with allergen-specific IgE-free serum showed that all isotypes recognized the major epitopes seen by IgE. These results suggest that there may be a "selective competition" among isotypes for allergens that is driven by the ability to produce high affinity, allergen-specific immunoglobulins.