Caveolae are 50-100 nm invaginations in the plasma membrane. Caveolae and the protein caveolin-1 (Cav1) have been shown to be important in many signaling pathways in different cell types; however, in some cell types caveolae and Cav1 do not seem to affect the investigated signaling pathways. In my thesis, I have investigated the role of caveolin-1 (Cav1) in metabolism and b3-adrenergic, LPA-, EGF- and PDGF-receptor signaling in brown adipocytes. Brown adipose tissue is responsible for nonshivering thermogenesis. Recent studies have shown that not only infants but also adult man can have brown adipose tissue and that the presence is negatively correlated with both obesity and age. By understanding how signaling for proliferation and differentiation in brown adipocytes is regulated, it could be possible in the future to activate brown adipose tissue to combat obesity and the metabolic syndrome. In brown adipocytes, both epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were able to induce proliferation, which was dependent on Erk1/2 activation. However, EGF and PDGF utilized different pathways to activate Erk1/2, with EGF signaling partially occurring via a Src-pathway (not involving PI3K/PKC) and PDGF via a PI3K/PKC/Src-pathway. Furthermore, LPA receptors were able to activate Erk1/2 via two pathways, one Gi/PKC/Src-pathway and one PI3K-pathway. For these receptors, Cav1-ablation did not affect the agonist-induced Erk1/2 activation. Cav1 was, however, required for proper b3-adrenergic receptor (b3-AR) signaling to cAMP and for adenylyl cyclase activity. In Cav1-ablated mice, the adrenergic receptors are desensitized. However, this desensitization could be overcome physiologically, and the Cav1-ablated mice were therefore able to survive in prolonged cold by nonshivering thermogenesis. In conclusion, ablation of Cav1 affected certain signaling pathways in brown adipocytes, while other pathways were not affected or could be physiologically rescued. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.

The rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function.

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing ptrifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-CpPhC6H5) substituents have been identified as a new class of peroxisome proliferatoractivated receptor γ (PPARγ) agonists that exhibit antitumorigenic activity. In this study, the PPARγ-active compounds decreased HT-29, HCT-15, RKO, HCT116 and SW480 colon cancer cell survival and KU7 and 253JB-V33 bladder cancer cell survival. In HT- 29, HCT-15, SW480 and KU7 cells, the PPARγ agonists induced caveolin-1 expression and this induction was significantly downregulated after cotreatment with the PPARγ antagonist GW9662. Since overexpression of caveolin-1 is known to suppress cancer cell and tumor growth, the growth inhibitory effects of the DIM compounds in these cell lines are associated with PPARγ-dependent induction of caveolins. These PPARγ-active compounds did not induce caveolin-1 in HCT-116 cells. However, these compounds induced NSAID-activated gene-1 (NAG-1) and apoptosis in this cell line. This represents a novel receptor-independent pathway for C-DIM-induced growth inhibition and apoptosis in colon cancer cells. In SW480 colon cancer cells 2.5-7.5 μM C-DIMs induced caveolin-1 whereas high concentrations (10 μM) induced pro-apoptotic NAG-1 expression. In athymic nude mice bearing SW480 cell xenografts DIM-C-pPhC6H5 inhibited tumor growth and immunohistochemical staining of the tumors show induction of apoptosis and NAG-1 expression. Thus, the PPARγ-active compounds induce both receptor-dependent and-independent responses in SW480 cells which are separable over a narrow range of concentrations and this dual mechanism of action enhances their antiproliferative and anticancer activities. Similar results were obtained for another structural class of PPARγ agonists namely 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and the corresponding methyl (CDDO-Me) and imidazole (CDDO-Im) esters. Structure-activity studies show that 1,1-bis(3'-indolyl)-1-(psubstitutedphenyl) methanes containing p-trifluoromethyl (DIM-C-pPhCF3), hydrogen (DIM-C-pPh) and p-methoxy (DIM-C-pPhOCH3) substituents activate Nur77 and induce apoptosis in pancreatic, prostate, and breast cancer cell lines. Nur77 agonists activate the nuclear receptor, and downstream responses include decreased cell survival, induction of cell death pathways including tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and PARP cleavage. Nur77 agonists also inhibit tumor growth in vivo in athymic nude mice bearing Panc-28 cell xenografts.

No description available.

Caveolae are a type of invaginated membrane domain that has been shown to be involved in several disease states, including lipodystrophy, muscular dystrophies and cancer. Several of these diseases are caused by the lack of caveolae or caveolae-related signaling deficiencies in the tissues in which the caveolar domain are abundant such as lung, adipose, muscle and their related endothelial cells. Caveolae are formed through the assembly of the membrane inserted protein caveolin, cholesterol and the recently described family of cavin proteins, which together form the caveolae coat. The work in this thesis focuses on understanding the protein components and mechanisms that control the biogenesis and dynamics of caveolae. We have found that the protein EHD2 is an important regulator and stabilizer of the caveolar domain at the cell membrane. EHD2 is a dimeric ATPase known to oligomerize into ring-like structures around lipid membranes to control their shape. We have characterized the domain interactions involved in the specific targeting and assembly of this protein at caveolae. We propose a stringent regulatory mechanism for the assembly of EHD2 involving ATP binding and switching of the EH domain position to release the N-terminus and facilitate oligomerization in the presence of membrane species. We show that loss of EHD2 in cells results in hyper- dynamic caveolae and that caveolae stability at the membrane can be restored by reintroducing EHD2 into these cells. In a study of the protein cavin-3, which is known to be an integral component of the caveolar coat, we showed that this protein is targeted to caveolae via direct binding to the caveolar core protein caveolin1. Furthermore, we show that cavin-3 is enriched at deeply invaginated caveolae and regulate the duration time of caveolae at the cell surface. In combination with a biochemical and cellbiological approach, the advanced fluorescence microscopy techniques, like Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection microscopy (TIRF), combined with correlative Atomic Force Microscopy (AFM) have allowed us to characterize distinct caveolae-associated proteins and their respective functions at caveolae.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

Glucocorticoids (Gcs) are a commonly used drug to target the glucocorticoid receptor (GR). The GR has a myriad of cellular and physiological effects, however, Gcs are clinically used for the treatment of inflammatory conditions due to the potent anti-inflammatory actions of GR. The anti-inflammatory effects come with serious side effects e.g. metabolic disease. I examine the role of lipid rafts in modulating the anti-inflammatory actions of Gcs, and the role of circadian rhythms in the control of Gc side effects. I tested the role of caveolin-1 (Cav1), a constituent of membrane lipid rafts, and its role in Gc suppression of inflammation. Gene expression analysis of mouse lung tissue showed that genetic depletion of Cav1 (CAV1KO) results in increased transactivation of Gc target genes. The increased Gc action, however, does not result in an increased effect on suppression of inflammation in a model of innate immunity: aerosolised lipopolysaccharide (LPS) induced lung inflammation or in a model of adaptive immunity: Ovalbumin. CAV1KO mice were protected from LPS induced inflammation, despite increased cytokine production. This suggests a differential response to LPS in lung parenchyma and alveolar macrophages dependent on Cav1. CAV1KO results in a pro-inflammatory phenotype in macrophages, and the opposite in parenchymal tissue. These data suggest that while Cav1 is an upstream regulator of Gc response, it does not have a strong enough effect to alter the ability of GR to repress inflammation in vivo. Gc treatment results in a strong metabolic phenotype, with aberrant energy metabolism, insulin resistance and hepatosteaotosis, I investigated how this side effect interacts with circadian rhythms, another key determinant of energy metabolism. Using transcriptomics of whole lung and liver taken during the day or the night, I demonstrate that the metabolic actions of Gc in the liver can be temporally separated, whilst maintaining consistent anti-inflammatory actions in both liver and lung. This temporal gene regulation by Gc is controlled by REV-ERB, a rhythmically expressed, orphan nuclear receptor, part of the core clock machinery, via a direct interaction with GR at key regulatory DNA loci. Genetic deletion of REV-ERB protects mice from the hepatosteotosis associated with Gc treatment. Taken together, these data suggest that Gcs are regulated upstream of the receptor by the core consitutent of membrane lipid rafts; Cav1, which modulates the Gc response in vivo. Also, that the GR action can be controlled by dosing at different times of day, separating the detrimental metabolic effects of Gcs from the beneficial anti-inflammatory effects. This is enabled through a direct interaction between GR and REV-ERB at key gene regulatory sites.

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Yes / Objectives Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods P EDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell cocultures and matrigel assays were employed to assess angiogenesis. Results P EDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions O ur data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc. / NIHR CDF; EULAR ODP