Connective tissue growth factor (CTGF/CCN2) expression in quiescent hepatic stellate cells is inhibited by a Twist‐1‐miR‐214 axis (649.7)

Connective tissue growth factor (CTGF/CCN2) drives hepatic fibrosis in vivo by stimulating fibrogenic pathways in hepatic stellate cells (HSC). Under normal conditions, CTGF/CCN2 expression is very low in quiescent HSC but CTGF/CCN2 expression is dramatically increased as HSC become activated during liver injury and adopt a fibrogenic phenotype. We have investigated the molecular basis for the suppressed CTGF/CCN2 levels in quiescent HSC. CTGF/CCN2 up‐regulation in fibrotic or steatotic livers, or in culture‐activated or ethanol‐treated primary mouse HSC was associated with a reciprocal down‐regulation of microRNA‐214 (miR‐214). By using protector or reporter assays to investigate the activity of the 3’‐untranslated region (UTR) of CTGF/CCN2 mRNA, induction of CTGF/CCN2 expression in HSC by fibrosis‐inducing stimuli was due to reduced expression of miR‐214 which otherwise inhibited CTGF/CCN2 expression by directly binding to the CTGF/CCN2 3’‐UTR. In primary mouse HSC, miR‐214 was co‐expressed with the transcription factor Twist‐1 which is a predicted target of an E box in the miR‐214 promoter. Expression of miR‐214 and Twist‐1 in HSC was dose‐dependently inhibited by ethanol or culture‐induced activation in a manner reciprocal to that of CTGF/CCN2. Transfection of primary D6 HSC (high endogenous Twist‐1 levels) with Twist‐1 siRNA reduced expression of miR‐214, but increased CTGF/CCN2 production. An opposite effect was shown by transfecting P6 HSC (low endogenous Twist‐1 levels) with Twist‐1 expression plasmids. Twist‐1 stimulated luciferase activity in HSC transfected with a wild‐type miR‐214 promoter but not with a mutant miR‐214 promoter lacking the E‐box site. We conclude that CTGF/CCN2 expression in HSC is dynamically regulated by an inhibitory Twist‐1‐miR214 axis. This regulatory mechanism may have utility for therapeutic intervention in pathways of CTGF/CCN2‐dependent fibrosis. Grant Funding Source : NIH 5 R01 AA021276