Kisspeptins (Kp), products of the Kiss1 gene that act via Gpr54 to potently stimulate GnRH secretion, operate as mediators of other regulatory signals of the gonadotropic axis. Mouse models of Gpr54 and/or Kiss1 inactivation have been used to address the contribution of Kp in the central control of gonadotropin secretion; yet, phenotypic and hormonal differences have been detected among the transgenic lines available. We report here a series of neuroendocrine analyses in male mice of a novel Gpr54 knockout (KO) model, generated by heterozygous crossing of a loxP-Gpr54/Protamine-Cre double mutant line. Gpr54-null males showed severe hypogonadotropic hypogonadism but retained robust responsiveness to GnRH. Gonadotropic responses to the agonist of ionotropic glutamate receptors, N-methyl-d-aspartate, were attenuated, but persisted, in Gpr54-null mice. In contrast, LH secretion after activation of metabotropic glutamate receptors was totally preserved in the absence of Gpr54 signaling. Detectable, albeit reduced, LH responses were also observed in Gpr54 KO mice after intracerebroventricular administration of galanin-like peptide or RF9, putative antagonist of neuropeptide FF receptors for the mammalian ortholog of gonadotropin-inhibiting hormone. In contrast, the stimulatory effect of senktide, agonist of neurokinin B (NKB; cotransmitter of Kiss1 neurons), was totally abrogated in Gpr54 KO males. Lack of Kp signaling also eliminated feedback LH responses to testosterone withdrawal. However, residual but sustained increases of FSH were detected in gonadectomized Gpr54 KO males, in which testosterone replacement failed to fully suppress circulating FSH levels. In sum, our study provides novel evidence for the relative importance of Kp-dependent vs. -independent actions of several key regulators of GnRH secretion, such as glutamate, galanin-like peptide, and testosterone. In addition, our data document for the first time the indispensable role of Kp signaling in mediating the stimulatory effects of NKB on LH secretion, thus supporting the hypothesis that NKB actions on GnRH neurons are indirectly mediated via its ability to regulate Kiss1 neuronal output.

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Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia (P=0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival (P=0.0003) and a higher 5-year cumulative incidence of relapse (P=0.005), when compared with IKZF1-deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1, did not affect the outcome of IKZF1-deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1-deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1+/− mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1+/− displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function.

Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses.

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We evaluated the effects of mediators that can be produced by smooth muscle and endothelial cells on growth responses in isolated arteries. Segments of carotid and renal arteries, denuded of endothelium, were isolated from adult rats and studied during tissue culture in the presence of indomethacin. Three days of culture in the presence of serum stimulated DNA synthesis in the media. During long-term culture new layers of cells developed at the borders of the arterial segments. Medial DNA synthesis depended less on serum than extramedial cell proliferation. During moderate stimulation, basic fibroblast growth factor and endothelin-1 enhanced and interleukin-1 and transforming growth factor-/? reduced medial DNA synthesis, whereas insulin-like growth factor-1, platelet-derived growth factor AA, platelet-derived growth factor BB, and angiotensin II were without effect. Of these factors, only endothelin-1 stimulated extramedial cell proliferation. In addition, serum-stimulated but not basic fibroblast growth factor-stimulated medial DNA synthesis was M echanisms of arterial smooth muscle cell (ASMC) growth control have been studied primarily by cell culture and by ballooncatheter injury in experimental animals.14 These approaches indicate that vascular cells can produce peptidergic growth factors, cytokines, and vasoactive agents that can interfere with growth responses.59 This is not restricted to ASMCs but applies to endothelial cells as well. 1015 A role for autocrine and paracrine mediators in vascular growth responses depends not only on their production but also on the responsiveness of the cell types involved. 16 Many agents have been shown to promote proliferation of isolated ASMCs in vitro, 141721 but this does not necessarily predict their activity in the vascular wall. 22- 23 Several causes have been suggested as underlying the different effects of growth factors in isolated and intravascular ASMCs. These include differences in cell density, phenotype, and presence of extracellular matrix components 24 and the multifactorial nature of ASMC growth control in situ, including not only growth factors but also growth inhibitors. 2427 In general, analysis of the effects of exogenous agents and contributions of endogenous factors to growth responses in vivo are complicated by the lack of sufficient

Btg1 and Btg2 encode highly homologous proteins that are broadly expressed in different cell lineages, and have been implicated in different types of cancer. Btg1 and Btg2 have been shown to modulate the function of different transcriptional regulators, including Hox and Smad transcription factors. In this study, we examined the in vivo role of the mouse Btg1 and Btg2 genes in specifying the regional identity of the axial skeleton. Therefore, we examined the phenotype of Btg1 and Btg2 single knockout mice, as well as novel generated Btg1 -/-;Btg2 -/- double knockout mice, which were viable, but displayed a non-mendelian inheritance and smaller litter size. We observed both unique and overlapping phenotypes reminiscent of homeotic transformation along the anterior-posterior axis in the single and combined Btg1 and Btg2 knockout animals. Both Btg1 -/- and Btg2 -/- mice displayed partial posterior transformation of the seventh cervical vertebra, which was more pronounced in Btg1 -/-;Btg2 -/- mice, demonstrating that Btg1 and Btg2 act in synergy. Loss of Btg2, but not Btg1, was sufficient for complete posterior transformation of the thirteenth thoracic vertebra to the first lumbar vertebra. Moreover, Btg2 -/- animals displayed complete posterior transformation of the sixth lumbar vertebra to the first sacral vertebra, which was only partially present at a low frequency in Btg1 -/- mice. The Btg1 -/-;Btg2 -/- animals showed an even stronger phenotype, with L5 to S1 transformation. Together, these data show that both Btg1 and Btg2 are required for normal vertebral patterning of the axial skeleton, but each gene contributes differently in specifying the identity along the anterior-posterior axis of the skeleton.

Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation usually arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are frequently observed in ETV6-RUNX1-positive leukemia. Here we report that Btg1 deficiency enhances the self-renewal capacity of ETV6-RUNX1-positive mouse fetal liver-derived hematopoietic progenitors (FL-HPCs). Combined expression of the fusion protein and a loss of BTG1 drive upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53. Similarly, ectopic expression of BCL6 promotes the self-renewal and clonogenic replating capacity of FL-HPCs, by suppressing the expression of p19Arf and Tp53. Together these results identify BCL6 as a potential driver of ETV6-RUNX1-mediated leukemogenesis, which could involve loss of BTG1-dependent suppression of ETV6-RUNX1 function.