Regulation of differentiation and gene expression in epithelial cells by cues from the cellular environment

Abstract

In Part 1, a new model system of hepatocyte differentiation was characterized, and environmental factors influencing hepatic differentiation were investigated. Cell line L2039 was derived from E14 fetal mouse liver by transformation with temperature-sensitive SV-40 T antigen, which allows rapid expansion of homogeneous cell populations at 33{dollar}\sp\circ{dollar}C and differentiation at 39{dollar}\sp\circ{dollar}C. At 33{dollar}\sp\circ{dollar}C, L2039 cells have an epithelial morphology, a high nucleocytoplasmic ratio, and a phenotype between bile duct and hepatocyte, expressing albumin, {dollar}\alpha{dollar}-fetoprotein, glutamine synthetase, IGF II receptor, fibronectin, laminin, and cytokeratins 8 and 19. The presence of vimentin, cytokeratin 14, and oval cell antigens link L2039 cells to cell populations thought to contain liver progenitor cells. Serum-free media conditions and extracellular matrix requirements have been determined for L2039 growth and differentiation. Differentiation begins within two days of growth at 39{dollar}\sp\circ{dollar}C, when L2039 cells cease dividing, show a glucocorticoid-dependent up-regulation of liver-specific genes including albumin, {dollar}\alpha{dollar}-fetoprotein, transferrin and phosphoenolpyruvate carboxykinase, and assume a more hepatocyte-like morphology. With time in culture, an increasing number of cells express albumin, indicating a conversion from a pre-hepatocytic to a hepatocytic phenotype. L2039 differentiation at 39{dollar}\sp\circ{dollar}C is enhanced by several agents, including sodium butyrate and dibutyryl cAMP. The expression of the transcription factors HNF-3{dollar}\alpha{dollar}, HNF-3{dollar}\beta{dollar}, HNF-4, C/EBP{dollar}\alpha{dollar}, C/EBP{dollar}\beta{dollar}, C/EBP{dollar}\delta{dollar}, and c-fos was also determined. The up-regulation of liver-specific genes at 39{dollar}\sp\circ{dollar}C correlated with the up-regulation to different extents of HNF-3{dollar}\alpha{dollar}, HNF-3{dollar}\beta{dollar}, C/EBP{dollar}\alpha{dollar}, C/EBP{dollar}\beta{dollar}, C/EBP{dollar}\delta{dollar}, and c-fos. Furthermore, seven day cultures of L2039 cells at 39{dollar}\sp\circ{dollar}C revealed a sequential induction of C/EBP isoforms over time. In L2039 cells, C/EBP{dollar}\alpha{dollar} expression was not strongly linked to albumin expression or cessation of cell division. Culture on Matrigel{dollar}\sp{lcub}\rm TM{rcub}{dollar} revealed the potential for biliary epithelial differentiation. Thus, L2039 is a hepatic precursor cell line responsive to matrix and hormones, and represents a promising model for the analysis of liver differentiation.;In Part 2, the regulation of the 92 kD type IV collagenase by changes in cell shape was investigated. In MDCK cells, anti-E-cadherin antibodies alter cell shape by disrupting cell-cell contacts, while sodium butyrate causes a marked flattening and spreading of cells. The disruption of cell-cell contacts led to a faint expression of the 92 kD collagenase, an effect enhanced by sodium butyrate which by itself did not induce collagenase; stromelysin expression was not regulated in these conditions. Similar results were obtained with a colon adenocarcinoma cell line. Secreted collagenase activity was not altered in these conditions in either cell line. Of the growth factors and other agents examined for the ability to regulate secreted collagenase activity, only PMA was effective. Examination of cytoskeletal, extracellular matrix, and cell contact proteins in MDCK cells revealed a disruption of the actin network, tight junctions, and fibronectin deposition by anti E-cadherin antibodies, an altered distribution of actin and cytokeratins 8 and 14, and a reduction in levels of laminin and {dollar}\beta{dollar}1 integrin by sodium butyrate. Thus, the novel induction of collagenase expression in epithelial cells by disrupted cell-cell adhesion in the presence of sodium butyrate is associated with changes in cell shape and structure.