The regulation of cytoskeletal and liver-specific gene expression during liver regeneration and primary hepatocyte culture
Date
1989
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Abstract
The hepatocyte allows for the study of events involved with both cellular growth and differentiation. The focus of this dissertation is to determine what role(s) the extracellular matrix and expression of certain cytoskeletal genes play in the regulation of hepatocyte growth and the maintenance of a differentiated state.
Three beta-tubulin eDNA clones (RBT1,RBT2,RBT3) were isolated and their sequences and patterns of tissue expression determined. RBT1 and RBT2 were found to be neural specific and expressed primarily during early and late brain development, respectively. RBT3 was ubiquitously expressed in all tissues tested. Comparison of the 3'-untranslated regions within the rat and mouse beta-tubulin multigene family revealed extensive conservation, not previously observed.
The expression of several cytoskeletal and liver-specific genes was examined during liver regeneration and in hepatocyte cultures maintained in a hormonally-defined, serum-free medium and plated on two different matrices: rat tail collagen and the EHS matrix. During liver regeneration and in hepatocytes cultured on rat tail collagen, there was a dramatic increase in tubulin mRNA levels coincident with but not linked to DNA synthesis. The message levels for other cytoskeletal genes (e.g., actin, alpha-tubulin) similarly increased, while a decrease was observed in the mRNA levels of the liver-specific genes, serum albumin and alpha1inhibitor III. Hepatocytes cultured on the EHS matrix resulted in the maintenance of low levels of cytoskeletal gene expression and high levels of liver-specific gene expression, similar to that observed in the normal liver. Nuclear "run-on" assays suggest that the cytoskeletal genes were regulated primarily at the posttranscriptional level, while the liver-specific genes were controlled predominately at the transcriptional level. Results from subcellular fractionation and two-dimensional gel electrophoresis of 35s-labelled proteins paralleled the results seen at the mRNA level. Preliminary work suggests that microtubule organization may play a role in the expression of the liver-specific genes which encode secreted proteins. These studies, which compare hepatocytes cultured on collagen or the EHS matrix gel, reveal that both cell-cell and cell-matrix interactions play a major role in the maintenance of the differentiated phenotype in hepatocytes.
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Dissertation (Ph.D.)--Boston University
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