The inhibitor of DNA-binding (Id) genes encode a family of helix-loop-helix proteins lacking the basic DNA-binding domain. The Id proteins function as dominant negative factors by dimerizing with other transcription factors, inhibiting DNA binding and transcriptional activation. Four members of the Id family (Id1-4) have been identified in mammals, expressed in spatially and temporally restricted patterns in many developing organs and have been implicated in the regulation of cell proliferation, apoptosis, differentiation and migration. Both Id1 and Id3 are highly expressed in the lung mesenchyme during vascular development, suggesting that they play indispensable roles in endothelial morphogenesis. In agreement, we found that Id1-/-Id3-/- lungs exhibit defects in distal angiogenesis after prolonged culture and implantation in renal capsules. MMP-2 is significantly under-expressed in Id1-/-Id3-/- lung endothelial cells, suggesting its contribution to the phenotype. These findings indicate that Id proteins are key components of embryonic lung vascular development. In this study we also found that, upon bleomycin treatment, Id1 expression was upregulated in a number of lung cell types but predominantly in endothelial cells, as revealed by double immunolabeling and quantitative FACS sorting analysis. As the result of ablated Id1 function, bleomycin-injured Id1-/- lungs showed increased vascular permeability and endothelial apoptosis. Accordingly, bleomycin-treated Id1-/- lung microvascular endothelial cells also showed decreased survival in culture. We detected a decrease in the level of Bcl-2, a primary anti-apoptotic protein, in Id1-/- endothelial cells, suggesting that downregulated Bcl-2 may promote endothelial apoptosis in the lung. Therefore, we propose that Id1 plays an important role in promoting endothelial survival in the adult lung upon injury. In addition, mice lacking Id1 function displayed increased collagen accumulation and fibrogenesis after long-term bleomycin exposure, suggesting that Id1 upregulation in the endothelium and other lung cell types may play a critical role in lung homeostasis.

While localized tumor growth may cause organ dysfunction and even death, metastases cause the vast majority (~90%) of human cancer deaths. Both autocrine and paracrine transforming growth factor-beta (TGF-beta) have been linked to invasive and metastatic tumor growth. The mechanism whereby autocrine TGF-beta elicits tumorigenic effects was investigated. The metastatic breast cancer cells, MDA-MB-231, secrete TGF-beta and express both the urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) that are important for cancer metastatic growth. Whether autocrine TGF-beta promotes invasive growth of MDA-MB-231 cells through regulation of uPA and/or MMP-9 protein levels and/or activity was studied. Inhibition of autocrine TGF-beta signaling decreased MDA-MB-231 cell invasion and uPA secretion. Inhibition of uPA proteolytic activity decreased cell invasion to the similar extent. The Smad proteins are the intracellular mediators for the canonical TGF-beta signaling pathway. However, TGF-beta receptors may transduce signals through Smad-independent pathways. My study demonstrates that the self-sufficiency of promoting invasive potential of tumor cells is through enhanced uPA secretion by autocrine TGF-beta in a Smad-dependent manner. While autocrine TGF-beta signaling modulates uPA protein secretion, exogenous TGF-beta increased uPA mRNA expression through RNA stabilization, suggesting distinct post-transcriptional mechanisms for regulation of uPA by different magnitudes of TGF-beta stimulation. Smad4 is both a tumor promoter and a suppressor. We previously observed inverse protein expression of Smad4 and claudin-1 in intestinal epithelial and colorectal cancer (CRC) cells and tissues. Claudin-1 is a tight junction protein with potential of enhancing metastatic growth of CRC cells. Whether Smad4 may act as a tumor suppressor by inhibiting claudin-1 expression in CRC was studied. In the Smad4-deficient, claudin-1-positive HT29 and SW480 CRC cells, Smad4 expression specifically downregulated claudin-1 protein expression through possibly transcriptional suppression. Previous findings suggest TGF-beta signaling-independent functions of Smad4. My study demonstrates that the Smad4-mediated suppression of claudin-1 expression is independent of TGF-beta signaling in SW480 and HT29 cells. These findings suggest a novel mechanism underlying the Smad4 tumor suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-beta-independent manner in CRC cells.

Wnt/β‐catenin signaling plays a critical role in metazoan development, stem cell maintenance, and human disease. The multicomponent β‐catenin destruction complex maintains low cellular β‐catenin in the absence of a signal and becomes inhibited in the presence of a Wnt ligand, allowing β‐catenin levels to rise. We have identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling. We show pyrvinium binds CK1α, enhances kinase activity and CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to effects on Axin and β‐catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of Adenomatous Polyposis Coli or β‐catenin inhibits both Wnt signaling and proliferation. These findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling. We also performed biochemical studies that identified positive feedback within the β‐catenin destruction complex between essential components, Axin and GSK3. Theoretical modeling of this positive feedback loop predicts bistability in the activity of the β‐catenin destruction complex. Through single cell studies, we generated experimental evidence for our theoretical predictions. These findings elucidate molecular design features that convert gradients into discrete binary cell fate decisions. In conclusion, this work combines chemical studies and systems-level analysis to uncover novel mechanisms of regulating the Wnt/β‐catenin pathway. Our findings highlight new strategies for targeted therapeutics directed against the Wnt pathway.

<p>Dynein is a minus-end directed microtubule motor complex that is required for a diverse range of biological processes, from intracellular-cargo transport to cell migration. Dynein is regulated at multiple levels and its functions are dictated by its affinity for microtubules, motor action, associations with specific cargos, and subcellular localization.</p> <p>During <i>Drosophila</i> spermatogenesis, dynein localizes to the nuclear surface at entry into meiotic prophase where it plays an essential role in mediating nucleus-centrosome coupling. Dynein also localizes to the nuclear surface of early postmeiotic spermatids, where it maintains associations between the nucleus and basal body and controls the position of the nucleus during elongation. I have identified the conserved gene <i>asunder</i> (<i>asun</i>) as a novel regulator of dyneins localization to the nuclear surface during <i>Drosophila</i> spermatogenesis. In addition, I have found that the dynein regulatory factor, <i>Lis-1</i>, also plays an essential role in promoting the localization of dynein to the nuclear surface, and that it may cooperate with <i>asun</i> in this process.</p>

In humans, the proper growth and homeostasis of endocrine islets in the pancreas is of great medical importance, in that loss and dysfunction of islet cells result in Type 1 and Type 2 Diabetes Mellitus. One of the major goals of diabetic-related researches is to understand how islets are formed and matured during normal development, so as to aid functional islet production in vitro for transplantation-based diabetes therapies. It has been well established that a bHLH transcription factor Neurogenin3 (Neurog3 or Ngn3) plays essential roles in endocrine islet differentiation in mice. However, it remains unclear how Ngn3 levels are regulated in endocrine progenitors and how Ngn3 coordinates islet cell differentiation and function. Previously, a zinc-finger transcription factor Myt1 (Myelin transcription factor 1) was identified from a microarray-based study aiming for factors that are specifically enriched in Ngn3+ pancreatic endocrine progenitors. Here, we report that Myt1 and Ngn3 form a feed-forward expression loop to promote endocrine differentiation. Specifically, loss of Myt1 partially compromises endocrine differentiation and islet function in the mouse pancreas, demonstrating that Myt1 plays a role in the generation of fully functional islet cells. Furthermore, although Myt1 expression largely depends on Ngn3 activity, Myt1 can enhance Ngn3 expression, suggesting that Myt1 contributes to endocrine commitment through Ngn3. To this end, reduced Ngn3 production at per cell level significantly impairs endocrine differentiation and endocrine/exocrine allocation. Finally, we discovered a previously unsuspected role of Ngn3 in islet cells. Sustained Ngn3 expression in hormone expressing endocrine islet cells is required for islet maturation and function. These studies not only provide important information regarding the regulation of endocrine differentiation, but also open up new directions to improve islet function under diabetic conditions.

Coordinated movement depends on specific connections between neurons, yet mechanisms that govern synaptic specificity are poorly understood. The simple, well-defined motor circuit of the nematode, C. elegans provides an optimal model system to study how neurons choose synaptic partners. Here, interneurons AVA, AVD, and AVE synapse with VA and DA motor neurons to establish the backward motor circuit, whereas interneurons AVB and PVC make connections with VB and DB motor neurons in the forward circuit. Mutations in the UNC-4 homeodomain transcription factor miswire VA motor neurons with inputs normally reserved for VBs, thereby disrupting backward locomotion. The Miller lab has shown that UNC-4 functions in post-synaptic VA motor neurons to block expression of VB genes that specify these aberrant connections. One VB gene, the transcription factor, CEH-12/HB9, is required for miswiring posterior VA motor neurons. My dissertation addresses two important questions: (1) What mechanism limits ceh-12/HB9 expression to posterior VAs and (2) What additional unc-4 pathway genes regulate inputs onto anterior VAs? First, my results establish that a posterior source of the diffusible ligand, EGL-20/Wnt is required for expression of CEH-12/HB9 in VAs. Genetic results indicate that the frizzled receptors, MOM-5 and MIG-1, respond to EGL-20/Wnt and drive ceh-12 expression through a canonical Wnt pathway. This also revealed a parallel pathway, involving LIN-44/Wnt and LIN-17/Frz, which antagonizes ceh-12 expression and promotes the creation of VA-type inputs. We hypothesize that UNC-4 inhibits expression of MIG-1 and MOM-5 to prevent posterior VAs from responding to EGL-20/Wnt. UNC-4 effectively biases overall Wnt signaling to favor the LIN-44/LIN-17 pathway. Second, I utilized genetic screens designed to isolate mutants that function in parallel to ceh-12. This approach revealed 22 independent blr (backward locomotion restored) mutations that map to 16 genetic linkage groups. Detailed phenotypic characterization of six blr mutants confirmed at least three genetic loci, blr-1, blr-3, blr-15, function in parallel to ceh-12 to regulate the specificity of interneuron input to VAs. Future molecular identification of these unc-4 pathway genes should provide key insights into the mechanism of synaptic specificity.

The Wnt/beta-catenin signaling pathway is a well-conserved signal transduction pathway that is highly regulated during metazoan development and is associated with various human diseases. In the current model of Wnt/beta-catenin signaling, Wnt ligands bind to its receptors, Frizzled and LRP6 (low-density lipoprotein receptor-related protein 6). These receptors transmits an intracellular signal that ultimately increases steady-state levels of cytoplasmic beta-catenin by inhibiting the phosphorylation of beta-catenin by the kinase GSK3. Elevated beta-catenin translocates to the nucleus and induces Wnt target gene expression. A major question in the field is how a Wnt/beta-catenin signal is transmitted from the receptors to mediate transmission of the Wnt signal from the plasma membrane. To understand how signaling through LRP6 inhibits beta-catenin degradation we expressed and purified the LRP6 intracellular domain and found that it stimulated beta-catenin stabilization while also stimulating the degradation of the negative regulator, Axin. Through additional egg extract and biochemical reconstitution experiments we found that LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin. The Frizzled receptor is predicted to have seven transmembrane domains, a feature that is characteristic of G protein-coupled receptors. To identify a role for heterotrimeric G proteins in Wnt/beta-catenin signaling, we screened major families of recombinant G protein alpha subunits in our egg extract system and found that Galphao, Galphaq, Galphai2, and Gbetagamma inhibit β-catenin degradation. We find that Gbeta1gamma2 is required for and promotes LRP6 phosphorylation and activation by directly recruiting GSK3 to the membrane and enhancing its kinase activity. We propose that heterotrimeric G protein activation results in formation of free Gbetagamma and Galpha, which act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription. Together, these studies provide insight on the molecular mechanism of the early intracellular events of Wnt/beta-catenin signaling.

Epithelial cells lining the lumen of the small intestine, called enterocytes, possess an exquisitely ordered array of microvilli collectively referred to as the brush border (BB). Microvilli are actin-rich membrane protrusions that cover the apical surface of most epithelial cell types; within microvilli of the enterocyte BB, the motor protein myosin-1a forms an ordered ensemble of bridges that link the plasma membrane to the underlying polarized actin bundle. Despite being one of the most abundant proteins in the enterocyte BB, it is unclear if myosin-1a demonstrates motor activity in the context of the microvillus, and if so, what roles this activity would play. Here, we show in vitro that addition of ATP to isolated BBs induces a plus enddirected translation of apical membrane along microvillar actin bundles. Upon reaching microvillar tips, membrane is "shed" into solution in the form of small vesicles. Because this movement demonstrates the polarity, velocity, and nucleotide dependence expected for a Myo1a-driven process, and BBs lacking Myo1a fail to undergo membrane translation, we conclude that Myo1a powers this novel form of motility. In vivo, we find a morphologically similar type of vesicle release from the tips of enterocyte microvilli; these vesicles retain the right-side-out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myosin-1a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the regulation of this novel activity. Intriguingly, we find that vesicles interact with bacteria present in the intestinal lumen, suggesting a role for vesicle production in mucosal barrier function. Furthermore, vesicles are able to detoxify the bacterial compound lipopolysaccharide, protecting cultured epithelial cells from its potent pro-inflammatory effects. In combination, these data show that myosin-1a is mechanically active in the context of enterocyte microvilli, that this activity is required for the proper regulation of vesicle production from microvilli, and that vesicles released from enterocyte microvilli may help to protect the epithelium from the intestinal microbiota.

Bone morphogenetic protein (Bmp) signaling is critical for vascular development and homeostasis. Defects in this pathway lead to multiple vascular diseases, including Heritable Pulmonary Arterial Hypertension (HPAH), which is genetically linked to mutations in Bone Morphogenetic Protein Receptor Type 2 (BMPR2). All forms of PAH display structural remodeling of resistance-level pulmonary arteries, suggesting that defective Bmp signaling might underlie other forms of PAH, even in the absence of BMPR2 mutations. Therefore, we utilized a genetics-based approach in mice to examine the functional role of Bmp signaling in hypoxia-induced pulmonary hypertension (PH).<p> Chapters 2 and 3 describe work that is now published. These studies illustrate that both Bmpr2 and Bmp2 (Bmp2+/-) mutant mice have defective regulation of pulmonary endothelial nitric oxide synthase (eNOS), indicating that Bmp signaling directly regulates pulmonary vascular tone. In contrast to Bmp2+/- mice, Bmp4 deficient (Bmp4LacZ/+) mice have preserved regulation of eNOS. Moreover, Bmp2+/- mice develop increased hypoxia-induced vascular remodeling and PH, while previous work showed that Bmp4LacZ/+ mice are partially protected from these effects. These studies indicate that Bmp2 and Bmp4 oppositely affect the development of hypoxic PH, and that regulation of eNOS is likely a key protective effect mediated by Bmp2 and Bmpr2 in the pulmonary vasculature.<p> The work shown in Chapter 4, which has been submitted for publication, explores the role of Id1 as a downstream mediator of Bmp4-dependent responses in the pulmonary vasculature. A previous study showed that Bmp4LacZ/+ mice display impaired hypoxia-induced vascular smooth muscle cell (VSMC) proliferation with decreased Id1 expression, suggesting that Id1 might promote VSMC proliferation in hypoxia. However, using Id1 null mice, we show that Id1 expression is not required for hypoxic-induced VSMC proliferation or PH. This finding might be due to functional compensation, since expression of the closely-related Id3 is selectively up-regulated in Id1 null peripheral vessel VSMC.<p> Collectively, these studies provide functional insight into Bmp signaling in pulmonary vascular homeostasis. They add to an understanding of human PAH by illustrating distinct downstream events associated with Bmp2- vs. Bmp4-signaling in vivo. Additionally, they provide potential targets for future therapies.

Focal adhesions (FAs) are sites at the interface between the cell and the ECM, linking integrin receptors and the actin cytoskeleton. In addition to serving as a structural platform, these sites are also robust sites of tyrosine phosphorylation and integrin signaling. When cells become adherent to the ECM, p130Cas (Crk-associated substrate) becomes tyrosine phosphorylated. Since p130Cas is primarily phosphorylated at tyrosines when it is localized to FAs, the localization of p130Cas to these sites appears critical to its ability to promote cell motility. The observation that with the exception of the SH3 domain, the C-terminus of p130Cas is the most highly conserved area of the protein, suggests an important role for this domain. Further observations that this domain has some sequence similarity to the FAK FAT domain is suggestive of this domain having an FA targeting function. The research in this dissertation aims to answer the following questions: 1) What contributions do the conserved N- and C-terminal domains make in the targeting of p130Cas to FAs and 2) What are the dynamics of p130Cas localization to FAs? In order to do so, fluorescently-tagged mutants of p130Cas were used to map the domain requirements for its FA localization. The localization of p130Cas to these sites was dependent on both the SH3 and CCH domains. The interaction of the SH3 domain with FAK was implicated as the major interaction mediating the localization of p130Cas through this domain. The SH3 and CCH domains were furthermore shown to be required for efficient p130Cas tyrosine phosphorylation to occur and the loss of tyrosine phosphorylation in deletion mutants was correlated with their inability to promote efficient cellular migration during wound-healing. Studies of the fluorescently-tagged p130Cas in live cells revealed that p130Cas localizes to FAs throughout their lifetime and exists in FAs with a high mobile fraction. Additionally, preliminary data suggested alternate sites of subcellular localization for p130Cas including filopodia and cell-cell contacts.