Jeffrey E. Kudlow - US grants

Prolactin secreting adenomata are a common cause of amenorrhea and infertility. tumors fortunately usually retain hormonal responsiveness such that their hormone production and growth can be controlled by the same hormones that modulate their normal counterparts. We have proposed that the growth of pituitary lactotrophs is regulated by growth factors whose expression in the pituitary gland is regulated hormonally. Previously we have localized transforming growth factor-alpha (TGFalpha) to lactotrophs and propose that this growth factor mediates the hormonal control of growth via the epidermal growth factor (EGF) receptor. Indeed, we have shown that estrogen-stimulated growth is preceded by an increase in TGFalpha mRNA in the pituitary while, bromergocryptine mediated growth inhibition is preceded by a decrease in TGFalpha mRNA. Our proposal is to enlarge upon these studies. Specifically, we propose to investigate the sites of TGFalpha expression in the pituitary and the modulation of its expression by estrogens and dopamine agonists using in situ hybridization. We also propose to develop transgenic mice in which TGFalpha overexpression is targeted to the lactotroph using the segments of the prolactin promoter which mediate- differing levels of tissue specific expression. These later studies will show whether TGFalpha stimulates lactotroph hyperplasia and hyperfunction. Finally, we have shown that TGFalpha expression is upregulated by phorbol esters and EGF. We have cloned 14 kb of the 5'-flanking region of the human TGFalpha gene of which a 1 kb proximal segment is capable of driving the expression of the luciferase reporter gene in MDA468 cells (human breast carcinoma cells). The 5'-promoter region of the TGFalpha gene is unusual in that it is G-C rich and does not contain typical TPA, EGF or estrogen response elements. On the other hand, it has striking similarity to the EGF receptor gene, which, in our systems, is coordinately regulated with the gene for its ligand, TGFalpha. We propose that these two genes use similar elements (cis- and trans-) to control their expression. Based on the structure of these genes, we believe these elements are distinct from the currently well characterized elements in other genes, raising the potential for the characterization of a novel transcription regulatory system. We plan to characterize the cis-acting elements in the TGFalpha gene that mediate phorbol ester and EGF responsiveness and to isolate and clone the proteins that interact with these elements. Together, the proposed studies will provide insight into the physiological role of TGFalpha in lactotroph, and form the basis for the understanding of its role and controlling mechanisms in other tissues.

The EGF system, perhaps in concert with other growth factor systems, plays a central role in human breast cancer. Levels of expression of the EGF receptor have been inversely correlated with the prognosis of the disease in humans and transgenic animals that overexpress one of the ligands for this receptor, TGFalpha, develop epithelial hyperplasia and carcinoma in the mammary gland. Estrogen regulation of breast cancer cell growth has also been partially attributed to the ability of this hormone to upregulate TGRalpha gene expression. Signaling through the EGF system may be under other complex controls. One such control could derive from regulation of the ability of the EGF receptor to signal in response to ligands. For some years, it has been appreciated that an alternate form of EGF receptor consisting of a soluble extracellular domain (ectodomain) is expressed by cancer cells and normal tissues. This segment of the receptor can bind ligand and may even be able to interact with the classical transmembrane receptor-tyrosine kinase to modulate ligand induced signal transduction. The ectodomain is produced as a result of alternate splicing of the EGF receptor transcript. We propose to study the EGF receptor ectodomain as a potential modulator of breast cancer growth and mammary gland development. We have produced milligram quantities of this molecule in a vaccinia virus system and have purified it to near homogeneity on a monoclonal antibody column. We will study the effect of this reagent on breast cancer cell growth in culture and correlate these effects with those on EGF receptor signal transduction. We will also study the effect of the ectodomain on the progression of human breast cancer in nude mice. Finally, we propose to create a transgenic mouse model in which overexpression of the ectodomain is targeted to the mammary gland using the MMTV-LTR as a promoter. These studies will form the basis for understanding the role of the EGF receptor signalling system in mammary gland development and breast cancer progression. This basic knowledge has the potential of developing into clinical applications for breast cancer and should be superior to the current protocols of using mouse derived monoclonal antibodies to the EGF receptor. This advantage relates to the observation that the EGF receptor ectodomain is a normal product of the EGF receptor gene.

Clinical studies and studies on isolated islets or Beta cell lines have indicated that chronic exposure of the Beta cell to supraphysiological levels of glucose results in impaired Beta cell function, an important component in the pathogenesis of type 2 diabetes. How glucose exerts this toxicity upon the Beta cell remains unclear. We propose that the glucose metabolite, glucosamine plays a role in Beta cell function and this glucose toxicity. We came to this hypothesis as a result of our observations on the mechanism of toxicity of the Beta cell- specific toxin, streptozotocin (STZ). STZ is chemically analogous to N- acetylglucosamine (GlcNAc). Furthermore, we found that the Beta cell contains approximately 100-fold more of the mRNA encoding the enzyme o- GlcNAc transferase (OGT). This enzyme modifies nuclear and cytoskeletal proteins by linking the monosaccharide GlcNAc to serine or threonine residues in the protein. The resulting O-GlcNAc modification appears to modify the activity of transcription factors. We found that STZ blocks the activity of an enzyme that removes o-GlcNAc from proteins. Treatment of rats with STZ results in the accumulation of the O-GlcNAc modification specifically in the pancreatic Beta cells, hours before Beta cell death. Because the Beta cells are so richly endowed with OGT, these cells may be the most susceptible to an accumulation of O-GlcNAc on intracellular proteins. We have also shown in other cell types, that nuclear O-GlcNAc is sensitive to ambient glucose concentrations. If this is also true in the Beta cell, then hyperglycemia and STZ may both increase Beta cell O-GlcNAc content, thereby leading to a common mechanism of Beta cell toxicity. The experiments proposed in this grant are designed to determine if hyperglycemia indeed result in increased Beta cell O-GlcNAc. We will also create transgenic mouse models in which glucosamine synthesis from glucose is either augmented or decreased. We will determine the effect of these alterations in glucosamine metabolism on Beta cell function. We have also found that cAMP-dependent protein kinase inhibits the enzyme responsible for glucosamine synthesis. We propose to study the mechanism by which glucosamine synthesis is inhibited. Together, these studies will establish the role of glucosamine in glucose toxicity on the Beta cell and a means of controlling glucose metabolism to glucosamine.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The laboratory has shown that protein modification with O-linked N-acetylglucosamine (O-GlcNAc) plays a direct role in the function of transcriptional activators and repressors. This modification, which results from glucose metabolism, also modulates the function of the proteasome, the major organelle involved in intracellular degradation of proteins. The chymotryptic activity of 26S proteasomes, but not 20S proteasomes against 4 amino acid peptides (LLVY) is blocked by incubation of the proteasome with O-GlcNAc transferase (OGT). In addition, the ATPase activity of intact proteasomes is blocked by OGT. Physiologically inactivated proteasomes from NRK cells treated with high glucose or glucosamine can be reactivated by recombinant O-GlcNAcase, the enzyme that removes this modification. Labeling studies on purified proteasomes with [3H]-GlcNAc indicate that the modified protein(s) have a molecular mass of about 45 kDa and that this substrate resides in the 19S regulatory cap of the proteasome. Since the proteasome degrades pro-apoptotic factors such as p53 and many of its downstream targets, inhibition of proteasome function might lead to the accumulation of these factors with the induction of apoptosis. The chemotherapeutic agent and GlcNAc analog, streptozotocin, also induces apoptosis through its property as a non-competitive inhibitor of the O-GlcNAcase. The proposed studies are designed to determine the biochemical linkage between the O-GlcNAc pathway and the proteasome. The ability of O-GlcNAc to block proteasomal function may also couple glucose metabolism to amino acid release from muscle wasting. The specific aims are as follows: General goal: Determine the role of O-GlcNAc in proteasomal function. 1. Determine the effect of O-GlcNAc transferase (OGT) and O-GlcNAcase on proteasome function in vitro using these enzymes to reversibly modify proteins in the proteasome in vitro. 2. Identify proteasomeassociated protein(s) that contain the O-GlcNAc modification and regulate proteasome function in a reversible manner. 3. Determine how O-GlcNAcylation of the proteasome 19S regulatory subunit modifies the function of the proteasomal peptidase and ATPases. 4. Using transgenic mice, determine the effect of proteasome blockade in vivo on epithelial cell apoptosis and muscle protein wasting. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The enzyme, O-GlcNAc transferase (OGT) that modifies nucleocytoplasmic proteins with O-GlcNAc has recently been shown to be part of the co-repressor complex turning off the transcription of genes. OGT associates with mSinSA and, therefore, is involved with genes that bind nuclear hormone receptors when the hormones, like estrogen, are not present. By modifying transcription activators, OGT cooperates with histone deacetylase to shut off transcription at sites where its scaffold, mSin3A, is present. Bound to OGT in a saturable fashion is NCOAT (nuclear cytoplasmic O-GlcNAcase and acetyltransferase), a bifunctional enzyme with O-GlcNAcase and histone acetyltransferase activities. NCOAT, by residing in co-repression complexes, allows the gene to be turned on again when hormone becomes present by reversing the actions of OGT and histone deacetylase. An NCOAT peptide that interacts with OGT, but has no enzymatic activities, blocks the ability of estrogen to induce estrogen-dependent genes or mammary development in transgenic mice. Thus, NCOAT is downstream of hormone and requires activation to reverse the actions of histone deacetylase and OGT. The aims of this project are as follows: 1. Further characterize transgene mice to determine the role of NCOAT in mammary development and other estrogen actions in the mammary gland. 2. Find where and how NCOAT is modified to activate it and what enzymes are involved. 3. Use the OGT interaction of NCOAT to design peptide therapy for breast cancer. This translational therapy will be developed to treat human breast cancer whose growth has become hormone-independent. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The enzyme, O-GlcNAc transferase (OGT) that modifies nucleocytoplasmic proteins with O-GlcNAc has recently been shown to be part of the co-repressor complex turning off the transcription of genes. OGT associates with mSinSA and, therefore, is involved with genes that bind nuclear hormone receptors when the hormones, like estrogen, are not present. By modifying transcription activators, OGT cooperates with histone deacetylase to shut off transcription at sites where its scaffold, mSin3A, is present. Bound to OGT in a saturable fashion is NCOAT (nuclear cytoplasmic O-GlcNAcase and acetyltransferase), a bifunctional enzyme with O-GlcNAcase and histone acetyltransferase activities. NCOAT, by residing in co-repression complexes, allows the gene to be turned on again when hormone becomes present by reversing the actions of OGT and histone deacetylase. An NCOAT peptide that interacts with OGT, but has no enzymatic activities, blocks the ability of estrogen to induce estrogen-dependent genes or mammary development in transgenic mice. Thus, NCOAT is downstream of hormone and requires activation to reverse the actions of histone deacetylase and OGT. The aims of this project are as follows: 1. Further characterize transgene mice to determine the role of NCOAT in mammary development and other estrogen actions in the mammary gland. 2. Find where and how NCOAT is modified to activate it and what enzymes are involved. 3. Use the OGT interaction of NCOAT to design peptide therapy for breast cancer. This translational therapy will be developed to treat human breast cancer whose growth has become hormone-independent. [unreadable] [unreadable]