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Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein-RNA interactions in the 3'-UTR of the collagen alpha1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is alphaCP(2). Recombinant alphaCP(2) is sufficient for binding to the 3'-UTR of collagen alpha1(I). To characterize the binding affinity of and specificity for alphaCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen alpha1(I) as probe. The binding affinity of alphaCP(2) for the 3'-UTR sequence is approximately 2 nM in vitro and the wild-type 3' sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein-nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA-DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant alphaCP(2) to the wild-type 3' sequence, although the kinetics of binding were slower.

There are several independent metabolic steps that determine the level of a protein in eukaryotic cells. The steady-state level of the mRNA encoding the specific protein is determined by rate of transcription, percentage of transcripts that are ultimately processed and transported to the cytoplasm, and half-life of the mRNA in cytoplasm. The amount of protein that accumulates from a particular transcript is influenced not only by the amount of mRNA present in the cytoplasm but also by the rate of translation of the mRNA and stability of the protein product. There is compelling evidence that the steady-state level of many proteins is regulated at multiple steps, and when there is a large change in the amount of either mRNA or protein it is likely that multiple steps in the metabolism of the mRNA and protein have been altered. In the case of type I collagen production in the fibrotic liver, recent work has shown that there is regulation of multiple steps resulting in an approximately 70-fold increase in collagen production by the hepatic stellate cells. In addition to the well-documented relatively small effect on transcription, there are effects on processing/transport of the mRNA, translation of the mRNA, and stability of the mRNA. Large changes of protein levels are produced by altering the rates or efficiency of multiple steps. The molecular details of some of these posttranscriptional regulatory events are currently being elucidated. Here we review the various potential steps for regulation in the synthesis of a protein and discuss how the synthesis of type I collagen may be regulated in the fibrotic liver.

The regulation of collagen alpha1(I) expression in hepatic stellate cells (HSCs) occurs in a complex fashion that is just beginning to be determined. The presence of regulatory sequences in both the 5' and 3' regions of the mRNA appear to be critical to its regulation in HSCs and are involved in the increased expression of collagen in activated HSCs. The 3' UTR contains a C-rich site that binds alphaCP, a known RNA-binding protein that is responsible for the increased stability of the mRNA in activated HSCs. Given that alphaCP is present in both activated and quiescent HSCs, there must be a mechanism for modifying alpha(CP to bind to the RNA in activated but not quiescent HSCs. The 5' region contains an evolutionary conserved stem-loop region that encompasses the translation initiation codon. This stem-loop can bind protein(s) in activated HSCs in an RNA cap-dependent manner. Such binding, together with the binding of alphaCP to the 3' UTR, can facilitate translation of collagen alpha1(I) mRNA, resulting in increased mRNA steady-state levels and collagen synthesis. A role of alphaCP in activating translation initiation has also been demonstrated. These two mechanisms work together to upregulate collagen alpha1(I) production in activated but not quiescent HSCs.