Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen-and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/ progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.WNT/β-catenin signaling | paracrine signaling | tumor biology

Addiction to nicotine may result in molecular adaptations in the neurocircuitry of speci®c brain structures via changes in the cyclic AMP-responsive element binding protein (CREB)-dependent gene transcription program. We therefore investigated the effects of chronic nicotine exposure and its withdrawal on CREB and phosphorylated CREB (p-CREB) protein levels in the rat brain. We report here that chronic nicotine exposure (1-h withdrawal) had no effect on the expression of CREB and p-CREB in the rat cortex and amygdala. On the other hand, decreases in the expression of CREB protein and phosphorylation of CREB occur in the cingulate gyrus, and in the parietal and the piriform but not in the frontal cortex during nicotine withdrawal (18 h) after nicotine exposure. It was also observed that CREB and p-CREB protein levels were signi®cantly decreased in the medial and basolateral, but not in the central amygdala during nicotine withdrawal (18 h) after chronic nicotine exposure. Furthermore, it was found that nicotine withdrawal (18 h) after chronic nicotine exposure leads to decreased CRE-DNA binding without modulating cAMP-dependent protein kinase A activity in the cortex and the amygdala of rats. In addition, chronic nicotine treatment produced anxiolytic effects whereas nicotine withdrawal (18 h) produced anxiety in rats as measured by the elevated plus-maze test. These results provide the ®rst evidence that decreased CREB activity and/or expression in speci®c cortical and amygdaloid brain structures may be involved in the underlying molecular mechanisms of nicotine dependence.

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Because P and NP rats are genetically bred for high and low alcohol drinking behavior, respectively, these results suggest the possibility that decreased expression of CREB protein in the amygdala may be associated with the high alcohol drinking behavior of P rats.

To define the molecular mechanisms of abnormal hypothalamic pituitary adrenal (HPA) axis during ethanol dependence, we investigated the effect of chronic ethanol treatment (15 days) and its withdrawal (24 h) on the expression of glucocorticoid receptors (GRs) and glucocorticoid response element (GRE)-DNA binding in the rat brain. The effects of chronic mianserin [serotonin (5-HT) 2A/2C antagonist] treatment on these parameters in various brain structures of control diet-fed and ethanolfed rats were also investigated. It was found that ethanol treatment and withdrawal significantly decreased the GR protein levels in cortical (cingulate gyrus, frontal, parietal, and piriform cortex) and amygdaloid (central, medial, and basolateral) structures and paraventricular nucleus (PVN) of hypothalamus of rats. It was also observed that ethanol treatment produced significant reductions in GR protein levels in various hippocampal structures (CA1, CA2, CA3, and dentate gyrus), but these changes were normalized during ethanol withdrawal. Ethanol treatment also significantly decreased GRE-DNA binding in rat cortex and hippocampus, which remained decreased in the cortex but reverted to normal in hippocampus during ethanol withdrawal. Chronic mianserin (alone) treatment had no effect on cortical GRE-DNA binding and GR protein levels in cortical, amygdaloid, or PVN structures but significantly decreased the GR protein expression in various hippocampal structures and GRE-DNA binding in whole hippocampus. However, when administered concurrently with ethanol treatment, mianserin significantly antagonized the reductions in cortical GRE-DNA binding and in GR protein expression in cortical, PVN, and central, but not medial and basolateral, amygdaloid structures during ethanol withdrawal. On the other hand, mianserin treatment along with ethanol administration significantly decreased the hippocampal GRE-DNA binding and GR protein expression in various hippocampal structures during ethanol withdrawal. Furthermore, ethanol treatment and its withdrawal or mianserin treatment had no effect on the neuron-specific nuclear protein levels in the various brain structures. Taken together, these results indicate that interaction of 5-HT 2A/2C receptors with GRs in cortical, central amygdaloid, and PVN structures may play a role in neural mechanisms of alcohol dependence. It is possible that decreased GR expression in PVN may be responsible for the abnormal HPA axis during ethanol exposure and withdrawal.