Effects of the extracellular matrix on hepatocyte gene expression and its regulation of the low density lipoprotein receptor-related protein



Benn, Sally Jean

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University of Guelph


The extracellular matrix (ECM) is believed to be a critical regulator of cellular function in most tissues. The influences of the ECM in regulating expression of various hepatocyte genes in primary culture were determined. Rat hepatocytes were plated on collagen Type I, a matrix which supports cellular proliferation, or on Matrigel, which supports a non-proliferating, differentiated hepatocyte phenotype. Total cellular RNA was collected, and cellular mRNA levels were determined by northern blot analysis. These studies showed that mRNA levels were significantly higher on Matrigel at 48 h than on collagen Type I for cytochrome P450 2A3 (45.6 ± 5.6% and 20.1 ± 6.3%, respectively) and the low density lipoprotein receptor-related protein (LRP) (43.8 ± 6.1% and 16.5 ± 7.3%, respectively). A similar, but non-significant pattern was seen for syndecan-2 (syn-2). In contrast, the mRNA expression patterns for liver regeneration factor (LRF-1) and CCAAT enhancer binding protein delta (C/EBP[delta]) were relatively matrix independent. The differences in hepatocyte LRP mRNA expression on collagen Type I and Matrigel in vitro were attributed to a 2-fold longer LRP mRNA half-life. This suggested that LRP gene expression may be regulated by characteristics of the 3'UTR of LRP mRNA. To test this, the full-length LRP 3'UTR and three LRP 3'UTR deletion mutants (D1, D2, D3) were cloned downstream of the luciferase gene in a pGL3/CMV reporter plasmid. BNL1MEA.7R.1 murine liver cells were cotransfected with the various LRP 3'UTR constructs and a [beta]-galactosidase construct. The D2 (2.7 ± 0.4 h) and D3 (3.0 ± 1.0 h) LRP deletion mutant half-lives were found to be significantly lower (p < 0.05) than the full-length LRP 3'UTR half-life (5.2 ± 1.0 h). The D1 mutant increased luciferase by 10%, whereas D2 and D3 decreased the reporter value relative to the full-length 3'UTR. The 30% decrease in reporter value for the D3 mutant was significant (p < 0.05). In conclusion, these studies demonstrate that LRP expression is matrix-dependent and the observed differences in hepatocyte LRP gene expression in vitro on different culture matrices are due to changes in LRP mRNA stability. Furthermore, sequences in the 3'UTR, at least in part, regulate LRP mRNA decay.



Extracellular matrix, Regulator, Low density, Lipoprotein, Hepatocyte