Fibrinogen, the major structural protein of a blood clot, is synthesized in the liver and secreted in the bloodstream as a multitimer of three different subunits, designated Aalpha, Bbeta, and gamma. The subunits are encoded by separate genes that diverged from a common ancestral sequence very early in evolution. In spite of their distant structural relationship, the three fibrinogen subunit genes are regulated in a highly coordinated manner in response to physiological stimuli. As part of the """"""""acute-phase response"""""""", a complex physiological reaction to a variety of stresses and tissue injuries, production of fibrinogen is elevated by the adrenal steroid hormones, glucocorticoids. This proposal focuses on determining the molecular mechanisms by which glucocorticoid hormones regulate transcription of the fibrinogen subunit genes. For these investigations, we are using a liver cell culture system derived from the frog Xenopus Laevis. Fibrinogen synthesis in highly purified Xenopus hepatocytes is dramatically induced about 20-fold by glucocorticoids. Therefore, this experimentally-manipulable system is ideal for a detailed dissection of the multiple elements involved in hormone responsiveness. Our basic experimental strategy is to define regions of the fibrinogen genes important for transcription by purifying fibrinogen gene fragments, generating deletion mutations in this DNA, and assaying effects on transcription after introducing the DNA into cultured Xenopus hepatocytes. The powerful gene transfection approach combines precise mutagenesis in vitro with analysis of functional consequences of the alterations in living cells.
The specific aims outlined in this proposal are: 1) Isolation and characterization of fibrinogen gene upstream regulatory DNA and insertion into a transfection vector; 2) Introduction of the fibrinogen DNA into primary Xenopus liver cells by transfection and demonstration of glucocorticoid-inducible transcription; 3) Generation of deletion mutations and analysis of effects on transcription in transfected liver cells; 4) Identification of DNA sequences that bind the glucocorticoid receptor in vitro; and 5) Identification of regulatory DNA sequences that bind other nuclear proteins important for control of transcription. These experiments will reveal DNA sequences and protein factors critical for basal and glucocorticoid-induced expression of the fibrinogen genes. Our long-term goal is to understand how highly coordinated activation of these three independent genes is achieved, even though different combinations of transcription factors are used. It is very important to maintain balanced synthesis of fibrinogen since it plays a vital role in blood coagulation and wound healing. There are several pathological states in which fibrinogen production is abnormal. For example, fibrinogen levels are elevated in thrombosis and heart disease. Conversely, fibrinogen and other blood-clotting factors are often insufficient in newborn infants, particularly those born prematurely. Understanding the factors underlying regulation of fibrinogen gene transcription will aid in developing treatments for conditions marked by aberrant fibrinogen production. GRANT R01HL48268 A novel M/r 28kDa red cell transmembrane protein was found to have a strong homology with MIP, the volume regulatory channel of lens. A related 28kDa protein was also found in renal tubules. The long term objectives of this proposal are to characterize the basic biology of the 28kDa protein in red cells, other tissues, and to delineate molecular pathology of 28kDa in blood diseases and other clinical disorders. I. GENETIC STUDIES-cDNAs for adult and fetal red cell and renal tubule 28kDa proteins will be isolated and sequenced. 28kDa genomic organization will determined and alternate splice isoforms will be characterized. Related proteins will be sought in nonerythroid tissues. II. BIOCHEMICAL STRUCTURAL--The higher order molecular structure of 28kDa, posttranslational modifications, and ultrastructure will be defined. III. EXPRESSION ASSEMBLY, AND FUNCTION--Expression and assembly of 28kDa subunits will be studied during erythroid differentiation, in renal tubules, and during mouse fetal development. Functional studies will be undertaken to characterize the behavior of purified red cell 28kDa inserted into planar membranes and after expression in Xenopus oocytes. IV. CLINICAL STUDIES--Employing methods and knowledge developed in I-III, patient samples will be analyzed for congenital or acquired molecular defects in 28kDa. Particular attention will be paid to red cells after prolonged cold storage and from patients with congenital hemolytic anemias, dyserythropoiesis, nutritional anemias and after chemotherapy. Kidney and urine will be examined for 28kDa in certain renal diseases.
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