Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^

Role of T cell response during vaccine immunity to tuberculosis

Tuberculosis remains one of the leading causes of death in man due to a single infectious agent. An estimated one-third of the world's population is infected with the causative agent, Mycobacterium tuberculosis (Mtb), despite the availability of the widely used vaccine, BCG. BCG has significantly varying protection rates with the lowest level of protection seen with the most common form of TB, adult pulmonary TB. Thus, numerous studies are being conducted to develop a more efficient vaccine. The ideal candidate vaccine would possess the ability to induce a solid and strong Th1 response, as this is the subset of T cells primarily involved in clearance of the infection. A novel vaccine should also induce such a response that may be recalled and expanded upon subsequent infection. Our group has introduced a mutant of a virulent strain of Mtb which lacks a component of the immunogenic antigen 85 complex (Ag85). Our vaccine, ΔfbpA, does not secrete the fibronectin binding protein Ag85A, and this has shown to lead to its attenuation in both murine macrophages and mice. Previous studies have also proven that ΔfbpA is more protective in mice than BCG against virulent aerosol challenge with Mtb. This study addresses the mechanisms of protection observed with ΔfbpA by phenotyping responding T cells. We first evaluated the ability of dendritic cells to present the mycobacteria to naïve T cells, an in vitro mock of primary immunization. We also measured the response of primed T cells to macrophage-presented mycobacteria to interpret the possible response of a vaccinated host to a boost. We concluded that ΔfbpA can elicit a stronger Th1 response compared to BCG in vitro, and further observed that this enhanced response is at least partly due to the presence of proteins encoded by a region of the genome absent in all strains of BCG. Finally, we observed this heightened Th1 response in the mouse model after primary vaccination and a virulent aerosol challenge. The cytolytic T cell response was also measured after virulent challenge and was found to be superior in the ΔfbpA-treated group when compared to the BCG group. ^

Alternative carbon metabolism: Virulence and regulation in Candida albicans

The interaction between C. albicans and innate immune cells is a key determinant to disease progression. Transcriptional profiling showed that C. albicans responds to macrophage phagocytosis by inducing pathways required for alternative carbon metabolism (beta-oxidation, the glyoxylate cycle, and gluconeogenesis), suggesting these pathways are important for virulence of C. albicans. ^ We have shown that deleting key genes (FOX2, FBP1) in these pathways results in virulence defects in an in vivo mouse model for systemic infection. Like icl1Δ/Δ mutants, fbp1Δ/Δ mutants are severely attenuated and fox2Δ/Δ mutants are mildly but significantly attenuated, indicating that carbon starvation is a relevant stress in vivo. ^ However, fox2Δ/Δ mutants also had unexpected phenotypes on certain carbon sources, unlike the case in Saccharomyces cerevisiae, suggesting these pathways are regulated differently in C. albicans. To test this, we identified the C. albicans regulators of these pathways based on those from S. cerevisiae and Aspergillus nidulans. ^ C. albicans has a partly conserved framework, but lacks two regulators (Oaf1p, Pip2p) controlling peroxisome biogenesis and beta-oxidation genes in yeast. Instead, C. albicans has a homolog, CTF1, of the A. nidulans fatty acid catabolism regulators FarA and FarB. We have shown that CTF1 is needed for growth on oleate (like FarA and FarB), expression of beta-oxidation and glyoxylate cycle genes, and full virulence. No function for CTF1 has previously been identified in C. albicans. Our data demonstrate a role for alternative carbon metabolism in the virulence of C. albicans and suggest that the regulation of these pathways is a mixture of the filamentous fungi and budding yeast systems. ^

Role of ovarian cancer antigen CA125/MUC16 in development and cancer

Cancer antigen 125 (CA125) is a tumor antigen that is routinely used to monitor the disease progress and the outcome of treatment in ovarian cancer patients. Elevated serum levels of CA125 are detected in over 80% of epithelial ovarian cancer patients. CA125 is a high molecular weight (>1M Dalton) mucin-type glycoprotein encoded by the MUC16 gene on human chromosome 19. Although MUC16 has served as the best serum marker for monitoring growth of ovarian cancer, roles for MUC16 in normal physiology and ovarian cancer are largely unknown. To understand the biological functions of MUC16, I characterized a mouse Muc16 homolog on chromosome 9 by means of expression pattern profiling, phenotype analysis of Muc16 knockout mice, and in vitro and in vivo studies of Muc16 null transformed ovarian surface epithelial (OSE) cells. ^ The mouse Muc16 homolog shares a conserved genomic structure with human MUC16. In addition to being expressed in mouse ovarian cancer, mouse Muc16 mRNA and protein were expressed in the mesothelia covering the heart, lung, ovary, oviduct, spleen, testis, and uterus. The conserved genomic structure and expression pattern of mouse Muc16 to human MUC16 suggests that mouse Muc16 is the ortholog of human MUC16. To understand the biological functions of Muc16, I generated Muc16 knockout mice. Muc16 knockout mice were viable, fertile and normal by one year of age. However, between 18 and 24 months of age, Muc16 knockout mice developed various tissue abnormalities such as ovarian cysts and tumors of the liver and other peritoneal organs. To determine the role of MUC16 in ovarian cancer progression, I established Muc16 null transformed ovarian surface epithelial (OSE) cell lines, following the same method to develop mouse model of epithelial ovarian cancer (Orsulic et al., 2002). Loss of Muc16 did not affect cell morphology, cell proliferation rate, or tumorigenic potential. However, Muc16-null OSE cells showed decreased attachment to extracellular matrix proteins as well as to primary mouse peritoneal mesothelial cells. Peritoneal mesothelia are the most frequent implantation sites of ovarian cancer. Furthermore, a pilot transplantation assay suggests that Muc16 null transformed OSE cells formed less disseminated tumors in the peritoneal cavity compared to wild-type OSE cells. ^ In conclusion, these results demonstrate that MUC16 is not required for normal mouse development or reproduction, but plays important roles in tissue homeostasis, ovarian cancer cell adhesion and dissemination. This study provides the first in vivo evidence of the roles of MUC16 in development, as well as ovarian cancer progression and dissemination. These studies offer valuable insights into possible mechanisms of ovarian cancer development and potential molecular targets for ovarian cancer treatment. ^

Elucidating the IGFBP2 signaling pathway in glioma development and progression

Diffuse gliomas are highly lethal central nervous system malignancies which, unfortunately, are the most common primary brain tumor and also the least responsive to the very few therapeutic modalities currently available to treat them. IGFBP2 is a newly recognized oncogene that is operative in multiple cancer types, including glioma, and shows promise for a targeted therapeutic approach. Elevated IGFBP2 expression is present in high-grade glioma and correlates with poor survival. We have previously demonstrated that IGFBP2 induces glioma development and progression in a spontaneous glioma mouse model, which highlighted its significance and potential for future therapy. However, we did not yet know the key physiological pathways associated with this newly characterized oncogene. We first evaluated human glioma genomics data harnessed from the publicly available Rembrandt source to identify major pathways associated with IGFBP2 expression. Integrin and ILK, among other cell migration and invasion-related pathways, were the most prominently associated. We confirmed that these pathways are regulated by IGFBP2 in glioma cells lines, and demonstrated that 1) IGFBP2 activates integrin α5β1, leading to the activation of key pathways important in glioma; 2) IGFBP2 mediates cell migration pathways through ILK; and 3) IGFBP2 activates NF-kB via an integrin α5 interaction. We then sought to determine whether this was a physiologically active signaling pathway in vivo by assessing its ability to induce glioma progression in the RCAS/tv-a spontaneous glioma mouse model. We found that ILK is a key downstream mediator of IGFBP2 that is required for the induction of glioma progression. Most significantly, a genetic therapeutic approach revealed that perturbation of any point in the pathway thwarted tumor progression, providing strong evidence that targeting the key players could potentially produce a significant benefit for human glioma patients. The elucidation of this signaling pathway is a critical step, since efforts to create a small molecule drug targeting IGFBP2 have so far not been successful, but a number of inhibitors of the other pathway constituents, including ILK, integrin and NF-kB, have been developed.

Breast cancer risk with aromatase inhibition and phytoestrogen-free diet

In this thesis a mouse model was used to examine the effect of pubertal estrogen inhibition and a phytoestrogen-free diet on the development of mammary glands. The study question was does treatment with aromatase inhibitor during puberty increase susceptibility to breast cancer among cohorts that consumed a diet free of phytoestrogens. The study design consisted of a cohort of mice treated with aromatase inhibitor, letrozole, during puberty and a vehicular group that was used as a control. Both groups were fed a diet free of phytoestrogens from the time of weaning until sacrifice during adulthood. The study aimed to assess mammary gland development in terms of breast cancer risk. The methods employed in this research included morphological and histological analysis of mammary glands, as well as estradiol, RNA and protein analysis. The main finding of the study was that mice exposed to aromatase inhibitor during puberty developed mammary glands with specific characteristics suggestive of vulnerability to oncogenesis such as increased lateral branching, increased number of glands, increase ductal hyperplasia, and diminished expression of TGFβ and p27 protein levels. The conclusions suggest that puberty is a critical period in which the mammary gland is susceptible to environmental threats that may result in deleterious epigenetic effects leading to an increased breast cancer risk in adulthood. This study has several public health implications; the most significant is that environmental threats during puberty may result in adverse mammary gland development and that phytoestrogen sources in the diet are necessary for normal maturation of the mammary glands.^

DIRECT EFFECTS OF METFORMIN ON PI3K AND RAS SIGNALING IN ENDOMETRIAL CANCER

Metformin has antiproliferative effects through the activation of AMPK and has gained interest as an antineoplastic agent in several cancer types, although studies in endometrial cancer (EC) are limited. The aims of this project were to evaluate pathways targeted by metformin in EC, investigate mechanisms by which metformin exerts its antiproliferative effects, and explore rational combination therapies with other targeted agents. Three EC cell lines were used to evaluate metformin’s effect on cell proliferation, PI3K and Ras-MAPK signaling, and apoptosis. A xenograft mouse model was also used to evaluate the effects of metformin treatment on in vivo tumor growth. These preliminary studies demonstrated that K-Ras mutant cell lines exhibited a decreased proliferative rate, reduced tumor growth, and increased apoptosis in response to metformin compared to K-Ras wild-type cells. To test the hypothesis that mutant K-Ras may predict response to metformin, murine EC cells with loss of PTEN and expressing mutant K-RasG12D were transfected to re-express PTEN or have K-Ras silenced using siRNA. While PTEN expression did not alter response to metformin, cells in which K-Ras was silenced displayed reduced sensitivity to metformin. Mislocalization of K-Ras to the cytoplasm is associated with decreased signaling and induction of apoptosis. Metformin’s effect on K-Ras localization was analyzed by confocal microscopy in cells expressing oncogenic GFP-K-RasG12V. Metformin demonstrated concentration-dependent mislocalization of K-Ras to the cytoplasm. Mislocalization of K-Ras to the cytoplasm was confirmed in K-Ras mutant EC cells (Hec1A) by cell fractionation in response to metformin 1 and 5 mM (p=0.008 and p=0.004). This effect appears to be AMPK-independent as combined treatment with Compound C, an AMPK inhibitor, did not alter K-Ras localization. Furthermore, treatment of EC cells with metformin in combination with PI3K inhibitors resulted in a significant decrease in proliferation than either agent or metformin alone. While metformin exerts antineoplastic effects by activation of AMPK and decreased PI3K signaling, our data suggest that metformin may also disrupt localization of K-Ras and hence its signaling in an AMPK-independent manner. This has important implications in defining patients who may benefit from metformin in combination with other targeted agents, such as mTOR inhibitors.

Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.

THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER Midan Ai, Ph.D. Supervisory Professor: Zhen Fan, M.D. Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism needs to be determined. In my studies, I found that high expression of HER2 is correlated with high expression of Brk in breast cancer cell lines. Silencing HER2 expression via RNA interference in HER2 over-expressed breast cancer cells resulted in Brk protein decrease and overexpression of HER2 in HER2 low-expressed breast cancer cells up-regulated Brk expression. The mechanism study indicated that overexpression of HER2 increased Brk protein stability. Brk was degraded through a Ca2+-dependent protease pathway involving calpain and HER2 stimulated Brk expression via inhibiting calpain activity. Calpastatin is a calpain endogenous inhibitor and the calpain-calpastatin system has been implicated in a number of cell physiological functions. HER2 restrained calpain activation via up-regulating calpastatin expression and HER2 downstream signaling, MAPK pathway, was involved in the regulation. Furthermore, silencing of Brk expression by RNA interference in HER2-overexpressing breast cancer cells decreased HER2-mediated cell proliferation, survival, invasion/metastasis potential and increased cell sensitivity to HER2 kinase inhibitor, lapatinib, treatment, indicating that Brk plays important roles in regulating and mediating the oncogenic functions of HER2. The Stat3 pathway played important roles in Brk mediated cell survival and invasion/metastasis in the context of HER2-overexpressing breast cancer cells. However, transgenic mice with inducible expression of constitutively active Brk (CA) in the mammary epithelium failed to develop malignant change in the mammary glands after Brk induction for 15 months which indicated that expression of Brk protein alone was not sufficiently to induce spontaneous breast tumor. Bitransgenic mice with co-expression of HER2/neu and inducible expression of Brk in the mammary epithelium developed multifocal mammary tumors, but there were no significant difference in the tumor occurring time, tumor size, tumor weight and tumor multiplicity between the mouse group with co-expression of Brk and HER2/neu and the mouse group with HER2/neu expression only.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^