Differential Regulation of Liver-Specific and Ubiquitously-Expressed Genes in Primary Rat Hepatocytes by the Extracellular Matrix

Abstract

Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase and β-actin) increased. When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (an EHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression in primary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole or actinomycin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of primary hepatocytes by inducing the transcription of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.

Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte

“…It was reported previously that DNA synthesis was affected by ECM components in primary cultured hepatocytes [22,23]. An ECM complex, such as Engelbreth–Holm–Swarm mouse sarcoma (EHS gel), was used as the culture substrate to improve differentiated function of primary cultured hepatocytes [24,25]. However, the interaction between the hepatocytes and the EHS gel was not fully understood.…”

Molecular Mechanisms Underlying the Dedifferentiation Process of Isolated Hepatocytes and Their Cultures

“…Actin fibers cluster at these canalicular surfaces [407]. Co-cultivation with another cell type or extracellular matrix overlay can partially reverse the effects seen in low density cultures [418][419][420], and stimulate a repolarization of cultivated hepatocytes [336,421].…”

“…Restoration of cell polarity by overlaying collagen gels on hepatocyte monolayer cultures, and/or by promoting homophilic and heterophilic intercellular contacts and signaling, upregulates liver-specific functioning and gap junctional intercellular communcation, and stimulates the formation of structures resembling in vivo bile canaliculi for weeks and months [10,11,226,418,425]. Meanwhile, the mRNAs of ubiquitously-expressed genes such as GAPDH are destabilized [419]. Three-dimensional matrices often more effectively induce differentiated cell function than traditional two-dimensional substrates [10,11,422,426,427], suggesting that the enhanced differentiation function is associated with morphological changes, rather than receptormediated cell-matrix interactions [428].…”

Loss of integrin linked kinase from mouse hepatocytes in vitro and in vivo results in apoptosis and hepatitis†

“…This is particularly true for hepatocytes, the parenchymal cells of the liver. [1][2][3][4] In fact, hepatocytes in culture maintained in the absence of matrix rapidly lose patterns of hepatocyte-specific gene expression and characteristic cellular micro-architecture. Interestingly, however, when hydrated complex matrix preparations [Matrigel: a matrix extract from EngelbrethHolm-Swarm mouse sarcomas or Type I Collagen Gels] 5 are added over de-differentiated hepatocytes, differentiation is restored within 3 days.…”