Investigation of KIT gene mutations in women with 46,XX spontaneous premature ovarian failure

BACKGROUND: Spontaneous premature ovarian failure presents most commonly with secondary amenorrhea. Young women with the disorder are infertile and experience the symptoms and sequelae of estrogen deficiency. The mechanisms that give rise to spontaneous premature ovarian failure are largely unknown, but many reports suggest a genetic mechanism in some cases. The small family size associated with infertility makes genetic linkage analysis studies extremely difficult. Another approach that has proven successful has been to examine candidate genes based on known genetic phenotypes in other species. Studies in mice have demonstrated that c-kit, a transmembrane tyrosine kinase receptor, plays a critical role in gametogenesis. Here we test the hypothesis that human KIT mutations might be a cause of spontaneous premature ovarian failure. METHODS AND RESULTS: We examined 42 women with spontaneous premature ovarian failure and found partial X monosomy in two of them. In the remaining 40 women with known 46,XX spontaneous premature ovarian failure we evaluated the entire coding region of the KIT gene. We did this using polymerase chain reaction based single-stranded conformational polymorphism analysis and DNA sequencing. We did not identify a single mutation that would alter the amino acid sequence of the c-KIT protein in any of 40 patients (upper 95% confidence limit is 7.2%). We found one silent mutation at codon 798 and two intronic polymorphisms. CONCLUSION: Mutations in the coding regions of the KIT gene appear not to be a common cause of 46,XX spontaneous premature ovarian failure in North American women

Proline-Rich Tyrosine Kinase 2 (Pyk2) Promotes Cell Motility of Hepatocellular Carcinoma through Induction of Epithelial to Mesenchymal Transition

Aims: Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells. Methodology/Principal Findings: We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization. Conclusion: These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT. © 2011 Sun et al.published_or_final_versio

Epithelial Hic-5/ARA55 expression contributes to prostate tumorigenesis and castrate responsiveness

Stromal–epithelial interactions dictate prostate tumorigenesis and response to castration. Hydrogen peroxide-inducible clone 5 (Hic-5/ARA55) is a transforming growth factor-beta (TGF-β)-induced coactivator of androgen receptor (AR) expressed in the prostate stroma. Interestingly, following castration, we identified epithelial expression of Hic-5/ARA55 in mouse and human prostate tissues. To determine the role of epithelial Hic-5 in prostate cancer progression and castration responsiveness, we compared LNCaP cells having Hic-5 stably expressed with the parental LNCaP cells following tissue recombination xenografts with mouse prostate stromal cells. We previously identified knocking out prostate stromal TGF-β signaling potentiated castrate-resistant prostate tumors, in a Wnt-dependent manner. The LNCaP chimeric tumors containing prostate fibroblasts conditionally knocked out for the TGF-β type II receptor (Tgfbr2-KO) resulted in larger, more invasive, and castration-resistant tumors compared those with floxed (control) stromal cells. However, the LNCaP-Hic5 associated with Tgfbr2-KO fibroblasts generated chimeric tumors with reduced tumor volume, lack of invasion and restored castration dependence. Neutralization of canonical Wnt signaling is shown to reduce prostate tumor size and restore regression following castration. Thus, we hypothesized that epithelial Hic-5/ARA55 expression negatively regulated Wnt signaling. The mechanism of the Hic-5/ARA55 effects on castration was determined by analysis of the c-myc promoter. C-myc luciferase reporter activity suggested Hic-5/ARA55 expression inhibited c-myc activity by β-catenin. Sequential ChIP analysis indicated β-catenin and T-cell-specific 4 (TCF4) bound the endogenous c-myc promoter in the absence of Hic-5 expression. However, the formation of a TCF4/Hic-5 repressor complex inhibited c-myc promoter activity, by excluding β-catenin binding with TCF4 on the promoter. The data indicate Hic-5/ARA55 expression in response to castration-enabled epithelial regression through the repression of c-myc gene at the chromatin level

Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner

The BMP Antagonist Follistatin-Like 1 Is Required for Skeletal and Lung Organogenesis

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development

A TSC22-like motif defines a novel antiapoptotic protein family

The apoptotic programme is evolutionarily conserved between yeast and metazoan organisms. We have previously identified a number of mammalian cDNAs capable of suppressing the deleterious effects of Bax expression in yeast. We herein report that one such suppressor, named Tsc22(86), represents the C-terminal 86 amino acids of the previously characterized leucine zipper (LZ) motif-containing transcriptional regulator Tsc22. Employing a genome-wide two-hybrid screen, functional genomics, and deletion mutagenesis approaches, we conclude that Tsc22(86)-mediated antiapoptosis is independent of the LZ motif and is likely independent of effects on gene transcription. Rather, a 16-residue sequence within the conserved 56-residue TSC22 domain is necessary for antiapoptosis. The presence of a similar sequence was used to predict an antiapoptotic role for two yeast proteins, Sno1p and Fyv10p. Overexpression and knock-out experiments were used to validate this prediction. These findings demonstrate the potential of studying heterologous proteins in yeast to uncover novel biological insights into the regulation of apoptosis