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

doi:10.1016/S0168-8278(99)80010-5

Journal of Hepatology

Volume 30, Issue 1, January 1999, Pages 77-87

https://doi.org/10.1016/S0168-8278(99)80010-5 Get rights and content

Abstract

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.

Section snippets

Transgenic mice

The TGFβ1-ko transgenic mouse line was developed and characterized previously (32). Heterozygous (+/−) mice were bred to produce homozygous (−/−) offspring. Wild type (+/+) litter mates served as controls. TGFβ1 genotyping was determined by polymerase chain reaction (PCR) analysis of tail DNA as described previously (33). All animal use was in full compliance with National Institutes of Health guidelines for humane care and approved by the University of North Carolina Institutional Animal Care

The effect of single-dose CCl₄ treatment on hepatic TGFβ mRNA of wt- and TGFβ1-ko mice

The mRNA levels of TGFβ1, -2, and -3 were determined by RT-PCR (Fig. 1). In a semi-quantitative assay the mRNAs of all three TGFβ isoforms were detectable in extracts of normal liver of wild-type (wt) mice. Wt (+/+) and TGFβ1-ko (−/−) mice had similar hepatic mRNA levels of TGFβ-2 and -3. As expected, TGFβ1 mRNA was not detected in the TGFβ1-ko mice (data not shown).

After 1 and 3 days post-CCl₄ treatment, wt mice had elevated TGFβ-1, -2, and -3 levels. TGFβ2 and TGFβ3 showed a similar profile,

Discussion

Our study was performed to investigate the causal role of TGFβ1 in the early activation of HSCs. We used two experimental approaches: 1) induction of an acute liver injury with single-dose CCl₄ in normal and TGFβ1-ko mice and 2) overexpression of TGFβ1 in the liver of wild-type mice using a recombinant adenovirus encoding human TGFβ1. This study revealed several findings that establish a critical role for TGFβ1 in accelerating and perpetuating the activation process of HSCs, but not as an

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

Abstract

Section snippets

Transgenic mice

The effect of single-dose CCl4 treatment on hepatic TGFβ mRNA of wt- and TGFβ1-ko mice

Discussion

Gastroenterology

J Biol Chem

J Hepatol

FEBS Lett

J Hepatol

Cytokine Growth Factor Review

Gene

J Mol Cell Cardiol

Hepatology

Anal Biochem

Hepatology

Methods Enzymol

Life Sci

J Biol Chem

J Biol Chem

Gastroenterology

J Biol Chem

J Surg Res

J Surg Res

Ito-cell gene expression and collagen regulation

Hepatology

Cellular distribution of transforming growth factor-β1 and procollagen types 1, III, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis

J Clin Invest

Modulation of collagen synthesis by fat-storing cells isolated from CCl4 or vitamin Atreated rats

Dig Dis Sci

Cellular sources of collagen and regulation of collagen production in liver

Semin Liver Dis

Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies

N Engl J Med

Mediators of hepatic fibrogenesis

Hepato-gastroenterology

Modulation of perisinusoidal cell cytoskeletal features during experimental hepatic fibrosis

Virchows Archiv B Cell Path

Sequential changes of extracellular matrix and proliferation of Ito cells with enhanced expression of desmin and actin in focal hepatic injury

Am J Pathol

Expression of the gene of the alpha-smooth muscle actin isoform in rat liver and in rat fat-storing (Ito) cells

Virchows Archiv B Cell Path

Purified rat liver fat storing cells divide in culture and contain collagen

Hepatology

Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture

Hepatology

Induction of platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture

J Clin Invest

Identification and characterization of a receptor for tissue ferritin on activated rat lipocytes

J Clin Invest

Cell-specific expression of transforming growth factor beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation

J Clin Invest

Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblast-like cells. A potential mechanism of self perpetuation in liver fibrogenesis

J Clin Invest

The effect of single-dose CCl₄ treatment on hepatic TGFβ mRNA of wt- and TGFβ1-ko mice

Modulation of collagen synthesis by fat-storing cells isolated from CCl₄ or vitamin Atreated rats