Zf9, a Kruppel-like transcription factor up-regulated in vivo during early hepatic fibrosis

Abstract

Wound repair in the liver induces altered gene expression in stellate cells (resident mesenchymal cells) in a process known as "activation." A zinc finger transcription factor cDNA, zf9, was cloned from rat stellate cells activated in vivo. Zf9 expression and biosynthesis are increased markedly in activated cells in vivo compared with cells from normal rats ("quiescent" cells). The factor is localized to the nucleus and the perinuclear zone in activated but not quiescent cells. Zf9 mRNA also is expressed widely in nonhepatic adult rat tissues and the fetal liver. The zf9 nucleotide sequence predicts a member of the Kruppel-like family with a unique N-terminal domain rich in serine-proline clusters and leucines. The human zf9 gene maps to chromosome 10P near the telomere. Zf9 binds specifically to a DNA oligonucleotide containing a GC box motif. The N-terminal domain of Zf9 (amino acids 1-201) is transactivating in the chimeric GAL4 hybrid system. In Drosophila schneider cells, full length Zf9 transactivates a reporter construct driven by the SV40 promoter/enhancer, which contains several GC boxes. A physiologic role for Zf9 is suggested by its transactivation of a collagen alpha1(I) promoter reporter. Transactivation of collagen alpha1(I) by Zf9 is context-dependent, occurring strongly in stellate cells, modestly in Hep G2 cells, and not at all in D. schneider cells. Our results suggest that Zf9 may be an important signal in hepatic stellate cell activation after liver injury.

Figure 1

Rat zf9 cDNA and deduced amino acid sequences of rat and human Zf9. The rat cDNA sequence is shown, beneath which are the deduced amino acid sequences of rat and human Zf9 (only those amino acids different from rat are shown for the human sequence). The Kozak consensus sequence just preceding the initiator methionine is indicated by a double underline. A potential endoplasmic reticulum targeting signal is boxed (amino acids 116–119). Potential O-linked glycosylation sites are indicated by amino acids with an asterisk. A potential Cdc2 kinase phosphorylation site is indicated by a double circle around serine 171. The C-terminal domain predicting three C2H2 zinc fingers is identified by a single underline.

Figure 2

(A) Induction of Zf9 during stellate cell activation in vivo; Western blot analysis of freshly isolated stellate cell lysates from a normal rat and a rat treated 3 hr earlier with CCl₄. A single protein of ≈46 kDa is identified. The blot was reprobed with anti-phosphatidylinositol 3-kinase. The induction after CCl₄ represents a 3-fold increase. A similar induction was reproducible in three experiments. (B) Increased biosynthesis of Zf9 during stellate cell activation in vivo. Pooled stellate cells from either two normal rats or two rats administered CCl₄ were plated in methionine-free medium containing [³⁵S]methionine and harvested at 45, 90, and 190 minutes or after 190 minutes with [³⁵S]methionine followed by a 20-hr chase in unlabeled medium. [³⁵S]Zf9 was purified by immunoaffinity chromatography by using a column containing anti-Zf9. SDS/PAGE autoradiography of eluted protein confirmed specific incorporation of [³⁵S]methionine into Zf9 (not shown). Data is depicted here as specific [³⁵S]methionine incorporation into protein from normal and activated stellate cells, normalized for cell number.

Figure 3

Induction of Zf9 and nuclear translocation during stellate cell activation in culture. (A) RNase protection in rat hepatic stellate cell isolates at intervals after plating on uncoated plastic. A 624-bp cRNA probe derived from the amino terminus (nucleotides 1–624) was used. The specific band is indicated by an arrow. Time 0 hr indicates mRNA derived from cells of a normal rat before plating. The 28S and 18S ribosomal RNA bands from the identical aliquots of RNA are indicated for each time point. Similar results were obtained in four different experiments. (B) Western blot of cells from the same isolate plated for increasing intervals on plastic identifies a single protein of ≈46 kDa, which is induced markedly within 3 hr of plating. (C) Immunofluorescence of rat stellate cells with anti-Zf9 antiserum is shown. (a) Freshly isolated cells after cytospin. Faint, patchy fluorescence is apparent surrounding cytoplasmic retinoid droplets, but nuclei are negative (white arrows). Cell borders cannot be appreciated. (×250.) (b) Cells maintained in culture on uncoated plastic for 14 days. There is marked nuclear and slight perinuclear fluorescence. Cells from the same isolate incubated with preimmune serum were completely negative (not shown). (×250.)

Figure 4

DNA binding by recombinant Zf9-GST. (A) Gel shift assays performed with recombinant full length Zf9-GST demonstrate strong specific binding to a consensus sequence for Sp1 but none to consensus oligonucleotides for Egr-1 or GATA-1. Nonspecific binding was minimized by inclusion of poly[d(I-C)], as described in Materials and Methods. (B) Gel-shift assay using recombinant full length Zf9-GST and an Sp1 consensus probe in which increments of anti-Zf9 antiserum were included led to the disappearance of a higher migrating protein–DNA complex (upper dot, left margin) and a faint intermediate migrating complex but no apparent supershift. A lower migrating complex was unchanged (lower dot, left margin). Similar results were obtained by using stellate cell nuclear extracts (not shown).

Figure 5

Transactivation by Zf9 following transient transfection. (A) Cotransfection of 500 ng of either full length Zf9 (Zf9-GAL4) or the 201-aa N-terminal domain (ZAD-GAL4) fused to the GAL4 DNA binding domain and a GAL4 responsive luciferase reporter results in equally strong transactivation in Hep 3B cells. (B) Cotransfection of 100- to 1,000-ng Zf9 (pAC-Zf9) and a 1-μg luciferase reporter containing the SV40 promoter/enhancer (pGL Control) in D. schneider S2 cells demonstrates transactivation by full length Zf9 but not by either the N-terminal 201-aa (ZAD) or the 82-aa zinc finger domain (DNA binding domain). Data represents mean of two experiments normalized to cell number.

Figure 6

Transactivation of the collagen α1(I) promoter by Zf9 in primary rat stellate cells and Hep G2 cells. Cotransfection of 0.5 μg Zf9 driven by the cytomegalovirus promoter (Zf9-pCIneo) and 1 μg of a collagen α(I) 1-luciferase promoter (pGL Col3) was performed in equal numbers of stellate cells from a normal rat 2 days after plating on plastic, and in Hep G2 cells. Although the fold activation was similar in the two cell types, the basal and Zf9-stimulated relative luciferase activities (shown in parentheses above each bar) were ≈12-fold greater in stellate cells. Data represents mean ± SD of sextuplicate values normalized for transfection efficiency. Similar results were obtained in four separate experiments.