Interactions between endothelin receptor B and transcription factors Sox10 and Pax3 in the melanocyte lineage

Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

Interactions between Endothelin Receptor B and Transcription Factors Sox10 and Pax3 in the Melanocyte Lineage

Genetic interactions that underlie developmental processes such as cell differentiation and pattern formation are complex and difficult to elucidate. Neural Crest (NC) cells and their derivatives offer an optimal system in which to probe for these complex interactions as they acquire different cell fates and constitute a variety of structures. The transcription factors Sox10 and Pax3 as well as the transmembrane receptor Endothelin receptor b (Ednrb) are temporally and spatially co-expressed early in NC cells and mutations in these genes lead to similar hypopigmentation phenotypes due to a reduced number of NC-derived melanocyte precursors, the melanoblasts. The goal of this study was to establish whether Sox10 and Ednrb or Pax3 and Ednrb interact to promote normal murine melanocyte development. Crosses of Sox10 or Pax3 with Ednrb heterozygous mutants showed that the double heterozygous hypopigmentation phenotype was significantly more pronounced than phenotypes of single heterozygotes, implying that a synergistic interaction exists between Sox10 and Ednrb and Pax3 and Ednrb. This interaction was further explored by the attempt to rescue the Sox10 and Pax3 hypopigmentation phenotypes by the transgenic addition of Ednrb to melanoblasts. Pigmentation was completely restored in the Sox10 and partially restored in the Pax3 mutant mice. The comparison of the number of melanoblasts in transgenic and non-transgenic Sox10 mutant embryos showed that the transgenic rescue occurred as early as E11.5, a critical time for melanoblast population expansion. Cell survival assays indicated that the rescue was not due to an effect of the transgene on melanoblast survival. A novel phenotype arose when studying the interaction between Ednrb and Pax3. Newborns appeared normal but by 3.5 weeks of age, the affected pups were smaller than normal littermates and developed a dome-shaped head; some also developed thoracic kyphosis. Affected pups were dead by 4 weeks of age: 80% were Pax3Sp/+ and 75% were female. When compared to normal littermates, affected mice had brains with enlarged 4th ventricles and more glia while skeletal staining showed kyphosis, wider rib cages and pelvic differences. An epistatic interaction resulting from the mixing of genetic backgrounds that is exacerbated in the presence of Pax3 heterozygosity is suspected.

Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

Endothelin receptor B and neural crest derived melanocyte development

Neural Crest cells (NCC) constitute a unique embryonic cell population that arises between the prospective epidermis and the dorsal aspect of the neural tube of vertebrates. NCC migrate ventromedially and dorsolaterally throughout the developing embryo giving rise to the peripheral nervous system constituents and melanocytes that ultimately reside in the skin and hair follicles respectively. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene manifest strikingly similar phenotypes characterized by hypopigmentation, hearing loss and megacolon these are due to absence of melanocytes in the skin and inner ear and lack of enteric ganglia in the distal part of the gut, respectively. Piebald lethal mice and humans with Hirschsprung's disease or Waardenburg syndrome carry different mutations in the Ednrb gene. The major goals of this project were to determine whether the action of Ednrb in NCC is required prior to commitment of these cells to the melanocytic lineage and to investigate its potential participation in the actual process of commitment. In order to achieve these goals transgenic mice that express Ednrb under two different regulatory elements were created. The first, Dct-Ednrb, expresses Ednrb under the control of the DOPAchrome tautomerase (Dct) promoter to direct expression to already committed melanocyte precursors. The second, Nes-Ednrb, expresses Ednrb under the regulation of the human nestin gene second enhancer to direct expression to pre-migratory NCC. Crosses of the Dct-Ednrb mouse with piebald lethal showed that the transgene was capable of rescuing the hypopigmentation phenotype of the later. This result indicates that the action of Ednrb after NCC commit to the melanocytic lineage is sufficient for normal melanocyte development. The Dct-Ednrb was further crossed with two other hypopigmentation mutants that carry mutations in the transcription factors Sox10 and Pax3. The transgene rescued the phenotype of the Sox10 mutant only. This suggests that Ednrb interacts with Sox10 but not with Pax3 during melanocyte development. The Nes-Ednrb mice developed a hypopigmentation phenotype that was augmented when crossed with piebald lethal or lethal spotting (mutation in Edn3, the ligand for Ednrb) mice but was rescued by over expression of Edn3. These results suggest that alterations in Ednrb expression early in development affect melanocyte development. This study provides novel information necessary to better understand the early embryonic development of NCC, clarifies specific interactions between different melanogenic genes and, could eventually help in the implementation of therapies for human pigmentary genetic disorders. ^

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Peroxisome Proliferator-Activated Receptor-γ Coactivator 1-α (PPARGC1A) Genetic Associations with Type 2 Diabetes in Three Ethnicities

Genetic heterogeneity, lifestyle factors, gene-gene or gene-environment interactions are the determinants of T2D which puts Hispanics and populations with African ancestry at higher risk of developing T2D. In this dissertation, the genetic associations of PPARGC1A polymorphisms with T2D and its related phenotypes (metabolic markers) in Haitian Americans (cases=110, controls=116), African Americans (cases=120, controls=124) and Cuban Americans (cases=160, controls=181) of South Florida were explored. Five single nucleotide polymorphisms of gene PPARGC1A were evaluated in each ethnicity for their disease association. In Haitian Americans, rs7656250 (OR= 0.22, pp=0.03) had significant protective association with T2D but had risk association in African Americans for rs7656250 (OR=1.02, p=0.96) and rs4235308 (OR=2.53, p=0.03). We found that in Haitian American females, both rs7656250 (OR=0.23, pp=0.03) had protective association with T2D. In African American females, rs7656250 (OR=1.14, p=0.78) had risk association whereas in males, it had significant protective effect (OR=0.37, p=0.04). However, the risk association exhibited by rs4235308 was stronger in African American females (OR=2.69, p=0.03) than males (OR=1.16, p=0.72). In Cuban Americans, only rs7656250 showed significant risk association with T2D (OR=6.87, p=0.02) which was stronger in females alone (OR=7.67, p=0.01). We also observed significant differences among correlations of PPARGC1A SNPs and T2D phenotypes. Positive correlation was observed for log Hs-CRP with rs3774907 (pp=0.03) in Cuban Americans respectively. Correlation of log A1C with rs7656250 (p=0.02) was positive in Cuban Americans while it was negative for rs3774907 in Haitian Americans (ppPPARGC1A correlations with T2D and its phenotypes among the three ethnicities studied (ii) the associations of PPARGC1A SNPs showed significant effect modification by sex. The findings suggest that variations in effects of PPARGC1A gene polymorphisms among three ethnicities and between sexes may have biomedical implications for the development of T2D as well as the phenotypes related to T2D.