Phosphorylation of estrogen receptor alpha, serine residue 305 enhances activity.

Upon ligand binding the estrogen receptor alters its conformation, dimerizes, binds to estrogen response elements (EREs), recruits cofactors and initiates the formation of a transcriptional complex. In addition to estradiol binding, hormone receptor activity is modulated by phosphorylation at several key residues. Previous studies have shown that p21-activated kinase-1 (Pak1) and cyclic-AMP dependent protein kinase (PKA) can phosphorylate ERalpha at serine residue 305. However, the effects of serine 305 phosphorylation on ERalpha activity have not been fully characterized. To study these effects, ERalpha S305E and S305A mutants were created to mimic constitutively phosphorylated or un-phosphorylated states, respectively. Using yeast two-hybrid assays we showed that dimerization of ERalpha S305E was still ligand dependent. However, the capability of dimerization in the presence of estradiol was significantly higher in S305E compared to wild-type ERalpha. Transactivation assays demonstrated that phospho-mimetic ERalpha S305E is active in the absence of ligand. Chromatin immunoprecipitation (ChIP) analysis shows a change of in vivo DNA binding in which S305E mutant binds to ERalpha DNA target sequences and exhibits increased residency in the absence of ligand. We also observed increased cell growth in cells stably transfected with S305E ERalpha. Thus, we suggest that phosphorylation of S305 does not trigger ERalpha dimerization but increases binding to target gene promoters, which can lead to increased cell growth in the absence of estradiol. This implies a shift from hormone-induced activation of ERalpha to activation through phosphorylation, which could confer resistance to hormone based therapies for breast cancer.

Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2(-/-) mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

Identification and characterization of novel human transcripts embedded within HMGA2 in t(12;14)(q15;q24.1) uterine leiomyoma.

The high mobility group A2 protein (HMGA2) has been implicated in the pathogenesis of mesenchymal tumors such as leiomyoma, lipoma and hamartoma. HMGA2 was pinpointed by mapping the breakpoints in the chromosomal translocations in 12q15, especially the t(12;14) that is commonly seen in uterine leiomyoma. It is generally assumed that altered expression of HMGA2 is an early event in the pathway to tumor formation. Here, we show evidence that three novel transcripts, A15, B6 and D12 are located within the HMGA2 gene itself and are transcribed from the opposite strand. These embedded transcripts are expressed at 6-20-fold higher levels in tumors compared to matched myometrium from the same patients. We estimate that the domain of increased expression extends 500kb on chromosome 12q15, and encompasses the majority of t(12;14) translocation breakpoints. However, a corresponding domain of consistently altered expression is not seen on chromosome 14 or outside of the chromosome 12 multiple aberration region. These data suggest that t(12;14) breakpoints contribute to the pathogenesis of uterine leiomyoma by interrupting a complex regulation of HMGA2 and other genes embedded within and around it. We also discovered a novel laminin receptor gene, transcribed from the opposite strand, within the promoter region of HMGA2. Although the roles for these embedded transcripts are still unknown, preliminary data suggest that they are members of the family of non-coding RNA and that they may play an important role in the pathology of uterine leiomyoma.

Tissue involution and the acute phase response.

After their roles in reproduction are completed, the mass of the uterus and the mammary gland decrease rapidly by the process of involution that involves an ordered series of events including apoptosis, neutrophil entry, the release of degradative enzymes, and phagocytosis of cellular debris. The acute phase proteins are produced by the liver and other tissues in response to inflammation or a toxic challenge. Uterocalin (SIP24/24p3) is one of these proteins. During involution, the mammary gland and uterus express high levels of uterocalin that reach an average of 0.2-0.5% of the total extractable protein at its peak. Uterocalin and its orthologues have been demonstrated in vitro to (1). bind certain fatty acids and (2). specifically induce apoptosis in neutrophils and other leukocytes. The period of uterocalin expression during involution is consistent with the hypothesis that one of its physiological roles is to induce apoptosis of invading neutrophils and delay the entry of neutrophils into the tissue until the second phase of involution. Interestingly, it has been shown that uterocalin expression remains higher in primiparous gland than in virgin glands after the pregnant glands have completely involuted. This observation and the known protective effect of early pregnancy on later development of breast cancer suggest that the ability of uterocalin to induce apoptosis in neutrophils might also decrease oxidative and carcinogenic activity in the gland and result in a lower mutation rate and thus a lower probability of cancer in the primiparous gland.