Bcr-Abl (break-point cluster region-abelson), the oncogenic trigger of chronic myelogenous leukemia (CML), has previously been shown to up-regulate the expression and activity of sphingomyelin synthase 1 (SMS1), which contributes to the proliferation of CML cells; however, the mechanism by which this increased expression of SMS1 is mediated remains unknown. In the current study, we show that Bcr-Abl enhances the expression of SMS1 via a 30-fold up-regulation of its transcription. Of most interest, the Bcr-Abl-regulated transcription of SMS1 is initiated from a novel transcription start site (TSS) that is just upstream of the open reading frame. This shift in TSS utilization generates an SMS1 mRNA with a substantially shorter 5' UTR compared with its canonical mRNA. This shorter 5' UTR imparts a 20-fold greater translational efficiency to SMS1 mRNA, which further contributes to the increase of its expression in CML cells. Therefore, our study demonstrates that Bcr-Abl increases SMS1 protein levels via 2 concerted mechanisms: up-regulation of transcription and enhanced translation as a result of the shift in TSS utilization. Remarkably, this is the first time that an oncogene-Bcr-Abl-has been demonstrated to drive such a mechanism that up-regulates the expression of a functionally important target gene, SMS1.-Moorthi, S., Burns, T. A., Yu, G.-Q., Luberto, C. Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation.

Hypothalamic kisspeptin neurons integrate and translate cues from the internal and external environments that regulate gonadotropin-releasing hormone (GnRH) secretion and maintain fertility in mammals. However, the intracellular signaling pathways utilized to translate such information into changes in kisspeptin expression, release, and ultimately activation of the kisspeptin-receptive GnRH network have not yet been identified. PI3K is an important signaling node common to many peripheral factors known to regulate kisspeptin expression and GnRH release. We investigated whether PI3K signaling regulates hypothalamic kisspeptin expression, pubertal development, and adult fertility in mice. We generated mice with a kisspeptin cell-specific deletion of the PI3K catalytic subunits p110α and p110β (kiss-p110α/β-KO). Using in situ hybridization, we examined Kiss1 mRNA expression in gonad-intact, gonadectomized (Gdx), and Gdx + steroid-replaced mice. Kiss1 cell number in the anteroventral periventricular hypothalamus (AVPV) was significantly reduced in intact females but not in males. In contrast, compared with WT and regardless of steroid hormone status, Kiss1 cell number was lower in the arcuate (ARC) of kiss-p110α/β-KO males, but it was unaffected in females. Both intact Kiss-p110α/β-KO males and females had reduced ARC kisspeptin-immunoreactive (IR) fibers compared with WT animals. Adult kiss-p110α/β-KO males had significantly lower circulating luteinizing hormone (LH) levels, whereas pubertal development and fertility were unaffected in males. Kiss-p110α/β-KO females exhibited a reduction in fertility despite normal pubertal development, LH levels, and estrous cyclicity. Our data show that PI3K signaling is important for the regulation of hypothalamic kisspeptin expression and contributes to normal fertility in females.

Kisspeptin-expressing neurons in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) of the hypothalamus relay hormonal and metabolic information to gonadotropin-releasing hormone neurons, which in turn regulate pituitary and gonadal function. Phosphatase and tensin homolog (PTEN) blocks phosphatidylinositol 3-kinase (PI3K), a signaling pathway utilized by peripheral factors to transmit their signals. However, whether PTEN signaling in kisspeptin neurons helps to integrate peripheral hormonal cues to regulate gonadotropin release is unknown. To address this question, we generated mice with a kisspeptin cell-specific deletion of Pten (Kiss-PTEN KO), and first assessed kisspeptin protein expression and gonadotropin release in these animals. Kiss-PTEN KO mice displayed a profound sex and region-specific kisspeptin neuron hyperthrophy. We detected both kisspeptin neuron hyperthrophy as well as increased kisspeptin fiber densities in the AVPV and ARC of Kiss-PTEN KO females and in the ARC of Kiss-PTEN KO males. Moreover, Kiss-PTEN KO mice showed a reduced gonadotropin release in response to gonadectomy. We also found a hyperactivation of mTOR, a downstream PI3K target and central regulator of cell metabolism, in the AVPV and ARC of Kiss-PTEN KO females but not males. Fasting, known to inhibit hypothalamic kisspeptin expression and luteinizing hormone levels, failed to induce these changes in Kiss-PTEN KO females. We conclude that PTEN signaling regulates kisspeptin protein synthesis in both sexes and that its role as a metabolic signaling molecule in kisspeptin neurons is sex-specific.

Galanin-like peptide (GALP) neurons participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signaling pathways utilized by both insulin and leptin. Therefore, we investigated whether PI3K signaling in neurons expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinizing hormone (LH) release. To this end, we deleted PI3K catalytic subunits, p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signaling in GALP neurons these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurons was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate if the GALP-neuron specific ablation of PI3K activity affected the susceptibility of the gonadotropic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. In contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to WT fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced MBH GALP expression in WT and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to WT, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and estrogen receptor co-localization within GALP neurons. Our data demonstrate that GALP neurons are direct targets of steroid hormones and that PI3K signaling regulates hypothalamic GALP mRNA expression and LH levels in a sex-specific fashion.