The aim of this research was to clarify the actions and interactions between factors implicated in the regulation of kit ligand (KITL) expression in rat granulosa cells by measuring Kitl1 and Kitl2 mRNA levels through quantitative PCR. Our findings included the observation that induction of the transcripts in primary granulosa cells by follicle-stimulating hormone (FSH), was not mimicked by dibutyryl cAMP and forskolin in spontaneously immortalized granulosa cells (SIGC). Transforming growth factor-beta1 (TGF-beta1) suppressed Kitl1 and Kitl2 mRNA levels in primary granulosa cells and SIGC - an effect mediated via Smad2 and Smad3. Exposure of primary granulosa cells to estrogen in vivo, via diethylstilbestrol (DES)-priming of immature rats and in vitro, by culturing in the presence of 17beta-estradiol, inhibited Kitl1 and Kitl2 mRNA expression, an effect mediated via TGF-beta1. Therefore, three levels of control are involved in regulating Kitl mRNA expression in the ovary: gonadotropins, local ovarian factors and steroid hormones.

The mutant epidermal growth factor receptor, EGFRvIII, is associated with tumour aggressiveness and drug resistance in glioblastoma. Our lab has shown that the molecular chaperone Hsp90 interacts with nascent EGFRvIII, and that EGFRvIII expression and function is dependent upon Hsp90 activity. In tumours, Hsp90 is found exclusively in an active form, where it is bound to cochaperones. One of these cochaperones, Cdc37, was found in the Hsp90/nascent EGFRvIII complex. Cdc37's role is to recruit certain kinase proteins to Hsp90 for folding. Its ability to bind these kinases is thought to be dependent upon phosphorylation of serine 13 in its client-binding domain by the kinase CK2. Here I have shown that Cdc37 is a limiting factor in EGFRvIII maturation, and its absence or inactivity may limit recruitment to Hsp90 and result in degradation. Using mutant constructs designed to mimic non-phosphorylated and phosphorylated Cdc37, I evaluated the role of Cdc37 phosphorylation in the stability of nascent EGFRvIII. Expression of the phospho-mimetic Cdc37 construct raised protein levels of nascent and mature EGFRvIII, while the non-phosphorylatable Cdc37 S13A mutant could not. Approximately 100 times more of the phospho-mimetic construct bound to nascent EGFRvIII than did the non-phosphorylatable form, and this increased Cdc37 binding was associated with more Hsp90 binding as well. Treatment of cells with the selective CK2 inhibitor, TBB, lowered the amount of Cdc37 and Hsp90 bound to nascent EGFRvIII, and reduced EGFRvIII protein levels. Taken together, these data indicate a positive role for Cdc37 and CK2 in EGFRvIII biogenesis, and suggest that Cdc37 phosphorylation may be a key factor in the recruitment of client to the Hsp90 complex.

The genes of the distal-less ( Dlx) gene family encode homeodomain transcription factors involved in the development of the brain, branchial arches, limbs, teeth, olfactory placodes and otic vesicles. In mice and humans, six Dlx genes have been identified and are organized in bigene clusters. The I56i cis-acting regulatory element is located within the intergenic region of the Dlx5/Dlx6 locus. This element has been shown to be active in the forebrain (telencephalon and diencephalon), the first and second branchial arches, and the apical ectodermal ridge. In this study, we identified eight putative transcription factor binding regions within the sequence of the I56i enhancer using DNaseI footprinting. These included putative binding sites for GATA-1, Engrailed-1, DLX and homeodomain transcription factors. Three to eight base-pair mutations were introduced into these putative binding sites. The effect of mutagenesis of four of these putative binding sites was evaluated in vivo in transgenic mice using a lacZ-beta-globin minimal promoter construct and in vitro using electrophoretic mobility shift assays (EMSA). Activity of the mutant enhancer in the forebrain of mouse embryos was reduced in some of the transgenic animals obtained. A reduction or complete loss of activity was seen in the branchial arches of all but one of the transgenic mice carrying the mutant enhancer constructs. EMSA demonstrated that proteins from the nuclear extracts of E11.5 mouse brains and E13.5 mouse forebrains bind fragments of DNA corresponding to the 156i enhancer sequences. Further, mutagenesis of the putative binding sites appears to reduce the ability of proteins to bind these areas of the I56i enhancer sequences. These experiments provide preliminary evidence suggesting that a number of factors may be involved in regulating the activity of the I56i enhancer and demonstrating the potential role of the I56i enhancer in the regulation of gene expression in the forebrain and branchial arches.

The Drosophila melanogaster model system is capable of providing valuable insight to neurodegenerative disease research. The Drosophila jing zinc finger transcription factor plays an essential role in CNS and tracheal cell differentiation. It is important to identify and characterize jing interactors to better understand its molecular mechanism. The JIGR1 gene was previously identified as a genetic modifier of jing and contains a MADF DNA-binding domain. The MADF domain is found in a large and diverse family with weak sequence homology. JIGR1 was found to genetically interact with other jing-interactors to maintain photoreceptor survival. JIGR1 is predominantly localized to the nuclei and is ubiquitously present throughout all stages of embryogenesis, particularly in the CNS and PNS, suggesting a role in fundamental cellular processes. Proper regulation of JIGR1 dosage is important, as its over-expression is associated with defects in the CNS and tracheal development. By characterizing JIGR1 and having a better concept of jing, with a homolog found in vertebrates, a better understanding of the complex vertebrate developmental pathways is gained.

It has previously been shown that high osmolarity is detrimental to cleavage-stage mouse embryo development in vitro, and that the presence of several organic osmolytes can provide protection against the detrimental effects of raised osmolarity. Whether the same is true for post-compaction stage embryos is unknown. In the present work, it was found that mouse post-compaction stage embryo development was inhibited by raised osmolarity. However, inhibition of embryo development from the 8-cell to the blastocyst stage occurred only at much higher osmolarities than that which inhibited development from the 1-cell stage. Glutamine, glycine, L-alanine and beta-alanine, which have been proven to function as organic osmolytes providing protection against increased osmolarity in cleavage-stage embryos (during the 1-cell through 4-cell stages), also protected post-compaction stage embryos from the detrimental effects of high osmolarity. Two other organic osmolytes, betaine and proline, which are effective in pre-compaction embryos, were not effective in providing protection against raised osmolarity in embryos developing in vitro from the 8-cell stage, nor were myo-inositol and taurine, which have been shown to be ineffective in cleavage-stage embryos. In addition, cleavage-stage embryos and post-compaction stage embryos were found to use different transport mechanisms to accumulate the four organic osmolytes that provided them with osmoprotection. When assessed in morulae, the amino acid transport system beta was found to be responsible for beta-alanine transport, while transport system B0+ mediated transport of glutamine, glycine and L-alanine. Glutamine, glycine, L-alanine and beta-alanine supported post-compaction stage embryo development, higher embryo cell number in blastocysts, and greater embryo volume at higher osmolarities. The four compounds identified do not share metabolic pathways or other such properties in common, and thus it is likely that post-compaction mouse embryos utilize them, in large part, as organic osmolytes.

Aortic carboxypeptidase-like protein (ACLP) is a 175kDa secreted protein that is downregulated with adipogenesis. I engineered 3T3-L1 preadipocytes that overexpressed ACLP protein and sustained the overexpression with differentiation. I assessed the role of sustained ACLP overexpression on 3T3-L1 adipogenesis, with a focus on possible ACLP interaction with collagen-I during differentiation. ACLP overexpression did not affect 3T3-L1 adipogenesis under standard cell culture conditions ACLP overexpression did not affect subconfluent preadipocyte proliferation, apoptosis in serum-supplemented preadipocytes, or serum-deprived preadipocyte cell death. Sustained ACLP overexpression did not affect collagen I/III protein expression, or the activity of matrix metalloproteinase (MMP)-2 and MMP-9 extracellular degrading enzymes. However, sustained ACLP overexpression on collagen-I coated dishes inhibited the expression of three key differentiation markers, peroxisome proliferator-activated receptor gamma, CCAAT enhancer-binding protein alpha and fatty acid synthase and decreased insulin-stimulated Akt/protein kinase B phosphorylation. This suggests that a collagen-I-rich environment is necessary for ACLP to alter preadipocyte function.

Formin proteins, characterized by two conserved formin homology (FH) domains, play an important role in cytoskeletal regulation and dynamics. The FH domains are most commonly implicated with the polymerization of F-actin and more recently with the stabilization of microtubules. Inverted formin 1 (INF1) is an atypical formin that has its FH1 and FH2 domains in its N-terminus. The C-terminus of INF1 contains five conserved regions unlike any other sequence found in the database. In this study, we demonstrate that INF1 discreetly associates with microtubules via a unique bipartite microtubule-binding domain (MTBD) in the C-terminus. Additionally, similar to the diaphanous related formin mDia, INF1 induces microtubule stabilization and acetylation. Expression of the MTBD alone, and not the FH2 domain, is sufficient to induce microtubule stabilization. However, in the absence of the MTBD, the N-terminus induces microtubule stabilization dependent on the presence of both the FH1 and the FH2 domains. In addition, knockdown of INF1 protein in NIH 3T3 fibroblasts results in a dramatic loss of acetylated microtubules suggesting INF1 is required for MT acetylation in these cells. Furthermore, we show that INF1 is not regulated by auto-inhibition, but is constitutively active both in vivo and in vitro. Our overexpression results suggest the INF1 C-terminus contains a putative regulatory domain targeted by an unknown inhibitory factor. Finally, we show that INF1 is capable of bundling both microtubules and F-actin simultaneously, implying that INF1 is a cytoskeletal cross-linking protein that could play an important role in cell polarity, cell migration and other cytoskeletal dependent processes.

Anaerobic metabolism as a consequence of low oxygen tension (hypoxia) is observed in various physiological and pathological conditions. Through the study of adaptive mechanisms to anaerobic metabolism, it was recently shown that an increase in the extracellular [H+] causes the relocalization and sequestration of the von Hippel-Lindau (VHL) tumor suppressor to the nucleolus. This results in an indirect increase in energy production through HIF transcription factor stabilization and a decrease in energy demand through silencing of ribosomal biogenesis. Mutagenesis of VHL revealed a pH-dependent nucleolar targeting sequence, NoDSH+. A bioinformatic search of this sequence identified proteins involved in major metabolic activities including: POLD1, the catalytic subunit of DNA polymerase delta; TAF1, subunit 1 of the general transcription factor TFIID; APC2, subunit of cell cycle protein APC/C; and UAP56, an mRNA splicing and export factor. Here we demonstrate that in response to acidic conditions, these proteins accumulate and become detained within the nucleolus. We also show that disruption of pH-dependent nucleolar sequestration of NoDSH+-containing proteins reduces cell viability through increasing cellular energy consumption. This data suggests that during anaerobic metabolism, cells utilize the nucleolus to sequester key proteins of basal metabolic processes, preventing their function, in order to maintain energy equilibrium by reducing metabolic demand.

MafK, a transcription factor that regulates beta-globin gene expression, undergoes subnuclear relocalization from heterochromatic to euchromatic regions during erythroid differentiation. As a first step to deciphering the role of MafK subnuclear relocalization, we investigated the characteristics of MafK heterochromatic localization before differentiation. Immunoprecipitation and mass spectrometry experiments indicate that MafK exists as a homodimer in the heterochromatic fraction, distinguishing it from MafK at the beta-globin locus. Immunofluorescence microscopy results demonstrate that a functional DNA-binding domain is necessary for MafK heterochromatic targeting. Native chromatin immunoprecipitation and quantitative PCR results suggest that MafK localizes to heterochromatin by binding pericentromeric major satellite DNA. A bioinformatics search for known Maf recognition elements indicates that pericentromeric heterochromatin contains novel MafK binding site(s). Our results support a role for subnuclear relocalization in regulating the spatial distribution and local concentration of MafK, presumably as an additional level of control over beta-globin gene expression, during erythroid differentiation.

La glycolyse anaérobique résulte en l'acidification du milieu extracellulaire et est associée à de multiples conditions physiologiques et pathologiques. La diminution du pH extracellulaire induit la détention de la protéine suppresseure de tumeurs VHL, une molécule impliquée dans la dégradation du facteur induit par l'hypoxie (HIF), dans le nucléole jusqu'au dégagement des protons. Ce processus est contrôlé par le signal de détention nucléolaire régulée par H+ (NoDS H+). Nous avons observé que le NoDSH+ de VHL possède des arginines méthylées à pH neutre qui diminuent l'affinité nucléolaire. Lors de l'accumulation d'ions H+ extracellulaires, la déméthylation des arginines du NoDSH+ induit la séquestration nucléolaire de VHL. Ce processus est réversé suite à la neutralisation du milieu acidique par la réméthylation des arginines et le relâchement de VHL du nucléole. Cette découverte est le premier exemple de la réversibilité de la méthylation des arginines régulée par une constante physiologique et contrôlant un processus cellulaire, la séquestration nucléolaire.