Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells.

Abstract

Background--Activation of hepatic stellate cells (HSCs) to a myofibroblastic phenotype is a key event in liver fibrosis. Identification of transcription factors with activities that are modulated during HSC activation will improve our understanding of the molecular events controlling HSC activation.

Aims--To determine if changes in E-box DNA binding activity occur during in vitro and in vivo activation of rat and human HSCs and to investigate mechanisms underlying any observed changes.

Methods--Nuclear extracts were prepared from rat HSCs isolated and cultured from normal and carbon tetrachloride injured rat livers and from HSCs isolated from human liver. EMSA analysis of E-box DNA binding activity was performed on nuclear extracts to determine changes during HSC activation. Western and northern blot analysis of MyoD and Id1 basic helix-loop-helix (bHLH) proteins was performed to confirm expression in HSC.

Results--HSC activation was associated with inducible expression of two low mobility E-box binding complexes that were immunoreactive with an anti-MyoD antibody. MyoD mRNA expression was found at similar levels in freshly isolated and activated HSCs; in contrast, MyoD protein expression was elevated in activated HSCs. Activation of rat HSCs was accompanied by reduced expression of the inhibitory bHLH protein Id1.

Conclusions--In vitro and in vivo activation of rat and human HSCs is accompanied by induction of MyoD binding to E-box DNA sequences which appears to be mechanistically associated with elevated MyoD protein expression and reduced expression of the inhibitory Id1 protein. Clarification of the role of MyoD and Id1 proteins in HSC activation and liver fibrogenesis is now required.

Keywords: liver fibrosis; hepatic stellate cell; basic helix-loop-helix transcription factors; MyoD; Id1

The ability of the hepatic stellate cell (HSC) to transdifferentiate into a proliferative a-smooth muscle actin ([alpha]-SMA) positive myofibroblast provides the liver with a mechanism for achieving wound healing.[1 2] However, excessive or progressive activation of HSCs can occur under conditions of alcohol abuse and viral infection and leads to accumulation of excess extracellular matrix in the form of scar tissue eventually resulting in cirrhosis.[1-5] It is therefore important to study the programme of molecular events that control HSC activation so as to understand how the processes of normal and pathological wound healing of the liver are controlled. We have previously used the cell culture model of HSC activation to describe the persistent induction of proteins belonging to the AP-1 and nuclear transcription factor [Kappa]B (NF[kappa]B) families which regulate transcription of important profibrogenic proteins such as tissue inhibitor of metalloproteinases 1 and intracellular adhesion molecule 1.[6 7] T his type of analysis can be applied to other transcription factors including those belonging to the basic helix-loop-helix (bHLH) family of proteins which have been shown to act as master transcriptional regulators of the growth and differentiation of mammalian cells.[8 10]

The DNA binding recognition sequence of bHLH proteins is the E-box (CANNTG) which is present in the promoter and enhancer regions of numerous developmentally regulated genes.[11 12] bHLH proteins exist as two distinct families, class A and B, that bind to the E-box in the form of homo- and heterodimers.[11-14] Class A bHLH proteins are often referred to as "E" proteins and are ubiquitously expressed. By contrast, class B proteins tend to be tissue or cell specific and include the myogenic transcription factors exemplified by the prototype factor MyoD which controls skeletal muscle differentiation.[9] A third set of related bHLH proteins known as inhibitors of differentiation (Id) lack a DNA binding domain and can act as inhibitors of the class A and class B proteins.[15] As it is conceivable that the dramatic phenotypic change associated with activation of HSC may be in part regulated by bHLH transcription factors, we have investigated changes in E-box DNA binding activity during in vitro and in vivo activat ion of rat and human HSCs.

Methods

ISOLATION AND CULTURE OF RAT AND HUMAN HSCs

Primary rat HSCs were isolated from the livers of normal or carbon tetrachloride treated six month old 500 g male Sprague-Dawley rats, as previously described. [16] Induction of acute liver damage in rats was achieved by administration of carbon tetrachloride by intraperitoneal injection with 0.2 m1/100 g sterile carbon tetrachloride in a 1:1 ratio with olive oil, as described previously. [17] Human HSCs were isolated from adult male human liver …