Arrested lung development in preterm infants leads to bronchopulmonary dysplasia (BPD). Inflammation and NF-κB activation in the fetal lung inhibit airway morphogenesis and contribute to BPD. The mesenchymal growth factor FGF-10 is crucial for normal airway branching and is decreased in lungs of patients with BPD. I therefore hypothesized that when activated, NF-κB disrupts normal FGF-10 transcription. I discovered that FGF-10 is in fact downregulated by NF-κB activation, but in an indirect manner. Further analysis of the FGF-10 promoter revealed many sites consistent with regulation by Sp proteins. Experiments revealed that Sp1 and Sp3 also regulate FGF-10; Sp1 activates transcription, while Sp3 downregulates Sp1-mediated expression. I further discovered that Sp3 and the NF-κB dimer interact during FGF-10 suppression, suggesting that NF-κB recruits Sp3 to the FGF-10 promoter. This is a novel mechanism of gene regulation in the developing lung.

I will introduce two novel genes isolated by my laboratory, hole and bves. This document will show that the benefits of gene discovery are invaluable. Bves, a novel family of cell adhesion molecules have been identified and my dissertation will be the first illustration of a function of this molecule in vivo. Data derived from this dissertation provide strong in vitro and in vivo evidence that bves plays an adhesive role in epithelial adhesion and morphogenetic movements during gastrulation in Xenopus and eye morphogenesis. Though gastrulation and eye development occur at different times during development and result in two very different structures, they serve as examples of a fundamental role Bves serves throughout embryogenesis. Xbves is expressed in a distinct group of epithelial and migrating cells in the Xenopus embryo. In vivo studies in the Xenopus embryo show that Xbves is required for proper migration of epithelial animal cells. The same migration defect is seen in an in vitro model of corneal epithelial cells. Combined with previous data that show bves participates in epithelial to mesenchymal transition in epicardium and coronary artery development, as well as data that show bves expression across many tissues, the data presented here support the idea that one gene product can participate in a variety of developmental processes. This study is an important contribution to understanding the overall larger concept of how bves functions in embryogenesis and the adult. Taken together, these studies have shown that Bves is important in proper epithelial cell migration and morphogenesis.

Peroxisome prolferator-activated receptor g (PPARg) is a member of the nuclear hormone receptor superfamily and is ligand activated by polyunsaturated fatty acids, certain arachadonic acid metabolites, and class of synthetic compounds with insulin sensitizing activity known as thiazoidinediones. PPARg is strongly expressed in the post-mitotic epithelial compartment of the normal human large intestine. Exposure of a panel of human colorectal cancer cell lines to PPARg agonists leads to a decrease in cell growth that is associated with a partial G1 arrest and increased levels of the cyclin dependent kinase inhibitor p21. A subset of these cells lines were resistant to the growth inhibitory effects of PPARg ligands. All four of the resistant lines contained a point mutation at codon 422 of the ligand binding domain of PPARg. Further studies suggested this mutation leads to a loss of functional PPARg due to a defect in the ability of the receptor to repress the transcrption of some target genes in the absence of exogneous ligand. Microrray technology was used to understand the genomic response of intestinal epithelial cells exposed to PPARg agonists. PPARg selective target genes included proteins linked to regulation of cell growth, colon epithelial cell maturation, and immune modulation. One of these genes, Transforming Growth Factor b Clone-22 (TSC-22), is a leucine zipper containing transcription factor that is capable of repressing gene transcription. Inhibition of TSC-22 using a dominant-negative construct partially blocked the ability of PPARg to induce growth arrest. In summary, these studies collective demonstrate that PPARg is a regulator of colorectal cancer cell growth and differentiation.

The ubiquitin-proteasome pathway (UPP) is the principal mechanism for the selective degradation of short-lived proteins. The proximal signal for UPP-mediated proteolysis is the covalent modification of target proteins with multiple ubiquitin polypeptides. Protein-ubiquitin conjugation is catalyzed by ubiquitinating enzymes, which assemble the polyubiquitin degradation signal on a target protein. It is postulated that removal of ubiquitin by deubiquitinating enzymes may also regulate protein targeting to the UPP. However, in comparison to ubiquitinating enzymes, relatively little is known about the functions or regulation of deubiquitinating enzymes. Dub-1 and Dub-2 are closely related deubiquitinating enzymes that were initially identified in hematopoietic cell lines as cytokine-inducible proteins. To gain insights into the substrate(s) and function(s) of the Dub enzymes, I examined the expression of Dub-1 and Dub-2 mRNA and protein, and investigated a potential role for these enzymes in regulation of signal transduction through the nuclear factor-kappa B (NF-kB) family of transcription factors. Expression analyses indicated that Dub-1 is expressed in the developing murine limb bud and in interleukin-3-stimulated FL5.12 pro-B cells. To investigate whether the principal inhibitory protein of NF-kB, IkBa, is a substrate for Dub-1 and Dub-2, a novel in vitro deubiquitination assay was established using polyubiquitinated IkBa as the substrate. In addition, I provide evidence for an IkBa-directed deubiquitinating activity in cytoplasmic lysates from a panel of cell lines. Using this and other complementary assays, I show that Dub-1 and Dub-2 do not deubiquitinate IkBa, do not stabilize IkBa, and do not modulate NF-kB activity. In addition, I show that Dub-1, but not the closely homologous Dub-2, is degraded via the UPP in HEK-293T cells. The UPP-mediated degradation of Dub-1 does not require an intact Dub-1 catalytic domain, thus indicating that this process does not proceed via Dub-1-catalyzed transfer of ubiquitin from a substrate to itself. Overall, these studies provide valuable insights as to the regulation of Dub-1 and Dub-2 that may help elucidate the substrate(s) and biological role(s) of these enzymes. Furthermore, the reagents generated for this dissertation will be useful for the study of Dub biochemistry and IkBa deubiquitination.

Cytokinetic actomyosin ring formation (CAR) in S. pombe requires two independent actin nucleation pathways, one dependent on the Arp2/3 complex and another involving the formin Cdc12p. Here we investigate the role of the PCH family protein, Cdc15p, in CAR assembly and find that it interacts with proteins from both of these nucleation pathways. Cdc15p binds directly to the Arp2/3 complex activator Myo1p, which likely explains why actin patches and the Arp2/3 complex fail to be medially recruited during mitosis in cdc15 mutants. Cdc15p also binds directly to Cdc12p. Cdc15p and Cdc12p not only display mutual dependence for CAR localization, but also exist together in a ring nucleating structure prior to CAR formation. The disruption of these interactions in cdc15 null cells is likely to be the reason for their complete lack of CARs. We propose a model in which Cdc15p plays a critical role in recruiting and coordinating the pathways essential for the assembly of medially located F-actin filaments and construction of the CAR.

Comparative genome analysis implied that a progressively more elaborate regulation of gene expression rather than invention of new genes is responsible for the organismal complexity, and that a constrained organization of metazoan enhancers is essential for the precise patterns of gene expression during development. This project was focused on studying of the transcriptional regulatory complex that directs tissue-specific gene expression and organ development in the prostate. Genetic manipulation of the rat probasin (Pbsn) gene promoter in transgenic mice uncovered a ~150 base pair core DNA fragment, which confers prostate-selectivity to the expression of this gene. Among a cluster of DNA sequence-specific transcription factors directly binding to this region, there are two cell-type limited proteins: the androgen receptor, a nuclear receptor, and Foxa1, a forkhead protein. Reporter assays indicated the forkhead response elements are crucial for AR-mediated transcriptional regulation. In vitro and in vivo binding assays established that the two proteins can bind adjacent DNA sequence concomitantly while the binding is not interdependent. A physical interaction occurs between Foxa1 and AR when the ligand for AR is present. This interaction is mediated through the forkhead domain and the AR DNA binding domain. Alignment of the forkhead and the androgen response elements resulted in the identification of a shared cis-regulatory code that is present in a variety of prostate-specific enhancers across species. The impact of Foxa1 loss-of-function and haploinsufficiency on mouse prostatic organogenesis was analyzed. Foxa1-/- prostates showed drastic morphologic alteration including a disorganized epithelial pattern resembling primitive epithelial cords, and an expansion in the mesenchymal smooth muscle layer. Cell type and ultrastructural studies demonstrated that Foxa1-/- epithelium is predominantly arrested as immature basal cells, consistent with a failure of luminal determination. Foxa1-/- basal cells actively express Sonic hedgehog (Shh), Ptc1 and Foxa2, proteins that are normally elevated during embryonic ductal budding in the UGS. Alteration of these signals correlates with the expansion of precursor cells and the modified epithelial-stromal pattern in Foxa1-/- prostates. In contrast, expression of the Nkx3.1 homeobox protein is absent in these Foxa1-/- cells. In addition a haploinsufficient phenotype was observed in heterozygous dorsal prostates showing a similar ductal morphologic defect. A novel Foxa1 target gene was identified. Upon examination of its promoter, critical forkhead response elements were identified to be immediately flanked by androgen response elements. We propose that Foxa1 regulates genes involved in prostatic ductal morphogenesis, and promotes epithelial cell maturation through balancing the effects of Shh, Foxa2, and Nkx3.1.

The NF-kappaB pathway has been shown to play a critical role in both adaptive and innate immunity and has been implicated as a focal point for induction of lung inflammation by a variety of inflammatory stimuli; however, the role of NF-kappaB in specific lung cell types remains unclear. We hypothesized that individual cell types in the lungs make important and unique contributions to the NF-kappaB dependent innate immune response. To determine the temporal and cell specific activation of NF-kappaB in vivo, an NF-kappaB reporter mouse in which expression of an enhanced green fluorescent protein (eGFP)/luciferase fusion protein cDNA driven by an NF-kappaB inducible promoter (NGL mouse) was generated. NF-kappaB activity was detected in intact, anesthetized animals by bioluminescence imaging and at the cellular level by detection of GFP on lung tissue sections. Using Eschericia coli lipopolysaccharide (LPS) and Pseudomonas aeruginosa models of lung inflammation, the timing and duration of NF-kappaB activation in alveolar macrophages, neutrophils, airway epithelium, and endothelium was determined to be dependent on the dose, duration, and route of delivery. To determine the role NF-kappaB in alveolar macrophages, bone marrow chimeras were generated by fetal liver transplantation (FLT) using the HLL transgenic NF-kappaB reporter mouse (HIV-LTR driving expression of luciferase) as the recipient and WT, IkappaB-alpha heterozygous, IkappaB-alpha knockout, and p50 knockout mice as donors. After LPS administration, lung NF-kappaB activation (bioluminescence imaging) and inflammation (neutrophil influx) were detected. IkappaB-alpha heterozygous FLT chimeras displayed enhanced and prolonged NF-kappaB activation and inflammation compared to WT FLT chimeras, while IkappaB-alpha knockout FLT and p50 knockout FLT chimeras displayed severe NF-kappaB activation and inflammation accompanied by mortality. To study macrophage/epithelium communication, co-culture studies using bone marrow derived macrophages from the above donors and epithelial cells containing an NF-kappaB reporter were performed. After LPS treatment, IkappaB-alpha knockout and p50 knockout macrophages demonstrated prolonged NF-kappaB activation, compared to WT, resulting in sustained NF-kappaB activation in epithelial cells. Taken together, these studies provide important insight into the importance of the NF-kappaB pathway in regulating lung inflammation and suggest IkappaB-alpha and p50 play a critical role in the active turn-off of NF-kappaB.

LIS1 and NudE(L) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Members of the LIS1 pathway play a critical role in fundamental cellular processes, such as differentiation, proliferation, and migration. Therefore, determining the precise nature of their actions will lead to significant insight into the complex process of embryogenesis. In this document, I present data revealing that cytLEK1, a large protein containing a spectrin repeat and multiple leucine zippers, is a novel component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. This is the first time that the cytLEK1 protein has been linked to a molecular pathway. I identified the exact binding domains in each molecule, and co-immunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, I was able to disrupt cytLEK1 function using a dominant negative approach, in addition to LEK1 knockdown, and thus examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of cellular morphology and microtubule organization. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. I also present data here examining, for the first time, the expression and localization of cytLEK1 in various cells and tissues. These experiments provide further evidence suggestive of the action of cytLEK1 as a regulator of the LIS1 pathway. Taken together, my studies reveal that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway and may play a critical role during murine development.

Hypoxia inducible vascular endothelial growth factor (VEGF) plays a major role in initiation and regulation of retinal neovascularization, which is the leading cause of severe vision loss and irreversible blindness in developed countries. Src family kinases (SFKs) are involved in a broad spectrum of cellular events. However, their roles in VEGF-mediated pathological retinal angiogenesis are completely unknown. My investigation showed that in vitro SFKs were essential for hypoxia-induced VEGF expression in retinal glial Müller cells, a major source of VEGF secretion during the pathogenesis of retinopathy, and for VEGF signaling in retinal microvascular endothelial cells (RMECs). However, neither process required phosphorylation of the SFK activation loop Tyr416. In addition, in RMECs, coexpressed SFK members Src, Fyn and Yes each displayed distinct properties in the regulation of VEGF-mediated cell events. All three kinases were required for VEGF mitogenic signaling. VEGF-induced cell migration was significantly increased in Fyn-deficient cells and decreased in Yes-deficient cells. Interference of Fyn, but not Src or Yes, impaired VEGF-induced tube formation in RMECs. In vivo, in a rat model of oxygen-induced retinopathy (OIR), I found that a significant increase of SFK Tyr416 phosphorylation was specifically associated with pathological retinal angiogenesis, but not with physiological intraretinal vascularizaion. Müller cells were the source of the elevated phospho-SFK Tyr416 signal. VEGF expression was also highly increased in these OIR retinas. Intravitreous injection of a selective SFK inhibitor (PP2) significantly reduced retinopathy. These findings indicate that aberrant SFK signaling may be an important factor in the pathogenesis of retinal neovascularization. Increased SFK activity or individual SFK member(s) are potential targets for therapeutic intervention in VEGF-mediated retinopathy.

Progression through the eukaryotic cell cycle involves the coordinated activation and inactivation of cyclin dependent kinases. One such family member, Cdk1p in a complex with Cyclin B, is activated at the G2/M transition in all eukaryotes and initiates the chain of events that ultimately lead to the phenotypic changes that occur during the progression through mitosis. The inactivation of Cdk1p-cyclin B at mitotic exit must be coordinated with respect to chromosome segregation to link nuclear and cytoplasmic divisions. This inactivation not only involves irreversible destruction of Cyclin B, but also a reversal of Cdk1p dependent phosphorylation events by the conserved Cdc14 family of protein phosphatases. We have identified the Cdc14 family member, Clp1p, in the fission yeast Schizosaccharomyces pombe, and studied how the disruption of Cdk1p phosphorylation events influences the coordinated inactivation of mitotic Cdk1p activity. Clp1p specifically disrupts the Cdk1p self-sustaining amplification loop involving Cdc25p activation and Wee1p inactivation at the exit from mitosis. This disruption is temporally regulated to occur only after Cdk1p activity wanes, involving a mechanism of direct inhibition of Clp1p phosphatase activity through Cdk1p dependent phosphorylation during early mitosis. Clp1p also participates in an auto-amplification loop during mitotic exit as it auto-catalytically reverses the inhibitory phosphorylation events to increase its activity, and prevents further inhibition of its activity by attenuating Cdk1p activity. Together, these findings point to a simple regulatory circuit that couples Cdk1p activation with its inactivation mediated through regulation of Clp1p phosphatase activity.