Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression

J. Armendariz-Borunda, C. P. Simkevich, N. Roy, Rajendra Raghow, Andrew Kang, J. M. Seyer

Research output: Contribution to journalArticle

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Abstract

Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Proα1(I) gene in FIC. LTIC expressed abundant transcripts of Proα1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Proα1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor β (TGFβ)-treated LTIC had a preferential increase in the rate of Proα1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGFβ treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGFβ. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGFβ-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGFβ-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.

Original languageEnglish (US)
Pages (from-to)817-824
Number of pages8
JournalBiochemical Journal
Volume304
Issue number3
StatePublished - Dec 1 1994
Externally publishedYes

Fingerprint

Replication Protein C
Hepatic Stellate Cells
Transcription Factor AP-1
Collagen Type I
Protein Binding
Gene expression
Carrier Proteins
Chemical activation
Gene Expression
Cultured Cells
Transforming Growth Factors
Genes
Transcription
Oligonucleotides
Amino Acid Motifs

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression. / Armendariz-Borunda, J.; Simkevich, C. P.; Roy, N.; Raghow, Rajendra; Kang, Andrew; Seyer, J. M.

In: Biochemical Journal, Vol. 304, No. 3, 01.12.1994, p. 817-824.

Research output: Contribution to journalArticle

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abstract = "Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Proα1(I) gene in FIC. LTIC expressed abundant transcripts of Proα1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Proα1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor β (TGFβ)-treated LTIC had a preferential increase in the rate of Proα1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGFβ treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGFβ. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGFβ-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGFβ-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.",
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