The role of TGFβ1 in initiating hepatic stellate cell activation in vivo

Background/Aims: The activation of hepatic stellate cells is a key initiating event in hepatic fibrogenesis. Although TGF beta 1 is a potent inducer of collagen alpha 1(I) expression in vitro and elevated levels of TGF beta 1 are found in patients and experimental animals with hepatic fibrosis and cirrhosis, the role of increased TGF beta 1 in the initiation of hepatic stellate cell activation in vivo is unknown. We used two experimental approaches to study this relationship: 1) Induction of an acute liver injury with carbon tetrachloride (CCl4) in normal and TGF beta 1-knockout (ko) mice, and 2) overexpression of TGF beta 1 in the liver of wild-type mice using a recombinant replication-deficient adenovirus encoding human TGF beta 1 (Ad-TGF beta 1). Methods: TGF beta 1-ko mice (n=6) and normal mice (n=6) were injected once intraperitoneally tip) with CCl4 (1 mu l/g BW) or mineral oil. Wild-type mice (n = 3) were injected intravenously with Ad-TGF beta 1 (10(10) pfu) or a control virus expressing beta-galactosidase (Ad-LacZ, 10(10) pfu), Animals were sacrificed after 3 days and total liver RNA was prepared, The expression of collagen alpha 1(I) mRNA normalized to GAPDH mRNA was measured by RNase protection assay, alpha-smooth muscle actin (alpha-sma) protein expression was analyzed by Western blotting. The expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs were determined semi-quantitatively with RT-PCR. Results: The collagen alpha 1(I) mRNA was increased 10-fold in CCl4-treated wild-type mice compared to the controls. This increase was reduced about 80% in the TGF beta 1-ko mice. The TGF beta 1 mRNA levels in the wild-type mice were proportional to the collagen alpha 1(I) mRNA levels. cr-sma, a marker of hepatic stellate cell activation, was expressed earlier and at a higher level in wild-type mice than TGF beta-ko mice after CCl4 treatment. The Ad-TGF beta 1 infected mice had 14-fold higher hepatic TGF beta protein levels and 15-fold higher collagen alpha 1(I) mRNA levels than the Ad-LacZ-infected control mice. Collagen alpha 1(I) mRNA levels were proportional to the transgenic TGF beta 1. mRNA levels, while the endogenous TGF beta 1 was only slightly higher than in the controls. TGF beta 2 and TGF beta 3 mRNA levels were elevated in CCl4-treated wild-type and TGF beta 1-ko mice and in Ad-TGF beta 1-infected mice compared to the controls. Conclusions: Absence of TGF beta 1 inhibits hepatic collagen alpha 1(I) mRNA and alpha-sma protein expression by the toxic stimulus CCl4, and targeted TGF beta 1 overexpression increases collagen alpha 1(I) mRNA and alpha-sma protein levels in the liver in vivo. Other TGF beta family members do not compensate for the TGF beta 1 deficiency. This indicates that TGF beta 1 accelerates, but is not absolutely required, for the activation of hepatic stellate cells.