Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.

PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype.It appears that PRL-1 is important in normal cellular growth control and could contribute to the tumorigenicity of some cancer cells.

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Liver regeneration in response to partial hepatectomy is a physiological growth response observed in the intact animal. Understanding the early signals that trigger liver regeneration is of vital importance to understand the liver's response to injury. It has been observed that several growth factors and cytokines, including epidermal growth factor (EGF) and interleukin-6 (IL-6), can activate members of the signal transducers and activators of transcription (Stat) family of transcription factors resulting in tyrosine phosphorylation of these factors, nuclear translocation, and an active DNA binding transcriptional complex. Because Stat3 participates in the regulation of primary growth response genes, we wondered if it is induced in the early phase of liver regeneration. We found that Stat3 DNA-binding activity is increased in the remnant liver within 30 minutes of partial hepatectomy and peaks at more than 30-fold at 3 hours. This induction is not observed after sham surgery. The induction of Stat3 appears to be part of the initial response of the remnant liver to partial hepatectomy, because it occurs in the presence of cycloheximide-mediated protein synthesis blockade. Activation of Stat3 is unusual, because it extends beyond the immediate-early time period and remains near peak level at 5 hours posthepatectomy. Although insulin-treated H35 cells activate many of the same immediate-early genes as regenerating liver, Stat3 is not induced in these cells. Because Stat factors are known to be inactivated by protein tyrosine phosphatases (PTPase), we showed that a PTPase is able to eliminate the DNA binding of hepatic Stat3.(ABSTRACT TRUNCATED AT 250 WORDS)

Class II transactivator (CIITA) is a global transcriptional coactivator of human leukocyte antigen-D (HLA-D) genes. CIITA contains motifs similar to guanosine triphosphate (GTP)-binding proteins. This report shows that CIITA binds GTP, and mutations in these motifs decrease its GTP-binding and transactivation activity. Substitution of these motifs with analogous sequences from Ras restores CIITA function. CIITA exhibits little GTPase activity, yet mutations in CIITA that confer GTPase activity reduce transcriptional activity. GTP binding by CIITA correlates with nuclear import. Thus, unlike other GTP-binding proteins, CIITA is involved in transcriptional activation that uses GTP binding to facilitate its own nuclear import.

The severe immunodeficiency type II bare lymphocyte syndrome (BLS) lacks class II MHC gene transcription. One defect from a complementation group A type II BLS patient is a 24 aa deletion in the MHC class II transactivator (CIITA). We show here that the molecular defect present in this protein is a failure of CIITA to undergo nuclear translocation. This defect was mapped to a position-dependent, novel nuclear localization sequence that cannot be functionally replaced by a classical NLS. Fusion of this 5 aa motif to an unrelated protein leads to nuclear translocation. Furthermore, this motif is not critical for transactivation function. This is a description of a genetic disease resulting from a novel defect in the subcellular localization of a transcriptional coactivator.

The liver is one of the few adult tissues that has the capacity to regenerate following hepatectomy or toxic damage. In examining the early growth response during hepatic regeneration, we found that a highly induced immediate-early gene in regenerating liver encodes RL/IF-1 (regenerating liver inhibitory factor) and is the rat homolog of human MAD-3 and probably of chicken pp4O. RL/IF-1 has IKB activity of broad specificity in that it inhibits the binding of p5O-p65 NF-KB, c-Rel-p50, and RelB-p50, but not p50 homodimeric NF-KB, to KB sites. Like RL/IF-1, several members of the NF-KB and rel family of transcription factors are immediate-early genes in regenerating liver and mitogen-treated cells. We examined changes in KB site binding activity during liver regeneration and discovered a rapidly induced novel KB site-binding complex designated PHF [posthepatectomy factor(s)]. PHF is induced over 1,000-fold within minutes posthepatectomy in a protein synthesisindependent manner, with peak activity at 30 min, and is not induced by sham operation. PHF is distinct from p5O-p65 NF-KB, which is present only in the inactive form in liver posthepatectomy. Although early PHF complexes do not interact strongly with anti-p50 antibodies, PHF complexes present later (3 to 5 h) posthepatectomy react strongly, suggesting that they contain a p50 NF-KB subunit. Unlike p5O-p65 NF-KB, c-Rel-p50, and RelB-p50 complexes, PHF (1, 9, 11, 21, 27-29, 32, 37, 38, 52, 58).We have isolated 41 novel immediate-early genes from regenerating liver and insulin-treated H35 cells and performed extensive analyses of immediate-early gene expression in regenerating liver, insulin-treated H35 cells, and serum-stimulated fibroblasts (37,38). We showed that the majority of immediate-early genes are expressed in all mitogen-treated cells, but about one-third show liver-specific induction. Subsequently, we found that one of the genes that * Corresponding author.we previously identified (37), RL-14, encodes a protein containing notchlike repeats. These repeats have been found in a variety of different proteins ranging from transcription factors to structural proteins (7,8,18,25,30,34,35,40,48,50,54). While we were performing the analysis of RL-14, very similar proteins, MAD-3 and pp4O, were identified and characterized as IKB-like in that they inhibited the binding of the NF-KB transcription factor to DNA (15,20).NF-KB, originally described as a B-cell-specific DNAbinding protein, is a ubiquitous transcription factor composed of p50 and p65 subunits that binds to KB sites found in the regulatory regions of many cellular genes involved in immune responses and/or acute-phase reactions like immunoglobulin K-chain genes, interleukin-2, beta interferon, and others (2,7,18,19,25,31 IKBot and IKB,(3,24,57). In pSO-p65 NF-KB, the binding site for IKB is likely to be on the p65 subunit, as IKB does not inhibit the binding of p50 homodimers to KB sites (4, 39). In cells, p5O-p65 NF-KB is normally present in an inactive, cytoplasmic form complexed with lKB. Activat...

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405–414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.