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 TGFβ1 is a potent inducer of collagen α1(I) expression in vitro and elevated levels of TGFβ1 are found in patients and experimental animals with hepatic fibrosis and cirrhosis, the role of increased TGFβ1 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 TGFβ1-knockout (ko) mice, and 2) overexpression of TGFβ1 in the liver of wild-type mice using a recombinant replication-deficient adenovirus encoding human TGFβ1 (Ad-TGFβ1). Methods: TGFβ1-ko mice (n=6) and normal mice (n=6) were injected once intraperitoneally (ip) with CCl4 (1 μl/g BW) or mineral oil. Wild-type mice (n=3) were injected intravenously with Ad-TGFβ1 (1010 pfu) or a control virus expressing β-galactosidase (Ad-LacZ, 1010 pfu). Animals were sacrificed after 3 days and total liver RNA was prepared. The expression of collagen α1(I) mRNA normalized to GAPDH mRNA was measured by RNase protection assay, α-smooth muscle actin (α-sma) protein expression was analyzed by Western blotting. The expression of TGFβ1, TGFβ2, and TGFβ3 mRNAs were determined semi-quantitatively with RT-PCR. Results: The collagen α1(I) mRNA was increased 10-fold in CCl4-treated wild-type mice compared to the controls. This increase was reduced about 80% in the TGFβ1-ko mice. The TGFβ1 mRNA levels in the wild-type mice were proportional to the collagen α1(I) mRNA levels. α-sma, a marker of hepatic stellate cell activation, was expressed earlier and at a higher level in wild-type mice than TGFβ-ko mice after CCl4 treatment. The Ad-TGFβ1 infected mice had 14-fold higher hepatic TGFβ protein levels and 15-fold higher collagen α1(I) mRNA levels than the Ad-LacZ-infected control mice. Collagen α1(I) mRNA levels were proportional to the transgenic TGFβ1 mRNA levels, while the endogenous TGFβ1 was only slightly higher than in the controls. TGFβ2 and TGFβ3 mRNA levels were elevated in CCl4-treated wild-type and TGFβ1-ko mice and in Ad-TGFβ1-infected mice compared to the controls. Conclusions: Absence of TGFβ1 inhibits hepatic collagen α1(I) mRNA and α-sma protein expression by the toxic stimulus CCl4, and targeted TGFβ1 overexpression increases collagen α1(I) mRNA and α-sma protein levels in the liver in vivo. Other TGFβ family members do not compensate for the TGFβ1 deficiency. This indicates that TGFβ1 accelerates, but is not absolutely required, for the activation of hepatic stellate cells.