Regulation of the human plasminogen activator inhibitor type 2 gene: cooperation of an upstream silencer and transactivator.

Transcriptional up-regulation of the plasminogen activator inhibitor type-2 (PAI-2) gene is a major response to cellular stress. The expression of PAI-2 is induced by a variety of cytokines and growth factors that act in a cell type- and differentiation stage-dependent manner. We previously reported that the human SERPINB2 gene promoter is controlled by three major transcription regulatory domains: an inducible proximal promoter, an upstream silencer (PAUSE-1), and a distal transactivator region between -5100 and -3300, which appears to overcome inhibition mediated by the silencer. The distal transactivator region is inducible by the phorbol ester PMA, a potent activator of the protein kinase C (PKC) pathway that is a powerful inducer of PAI-2 gene expression in monocytes, macrophages, and myelomonocytic cells as well as in epidermal keratinocytes. Here we show that a 21-bp region (-4952/-4932), containing an AP-1 element, is both necessary and sufficient for PMA-induced transactivator activity in PAI-2-expressing U937 cells. This site specifically binds FosB in PAI-2-expressing U937 cells but not in HeLa cells that do not express PAI-2, and overexpression of FosB, c-Fos, or c-Jun in HeLa cells is sufficient to cause derepression of transcription from the SERPINB2 promoter. Although FosB is likely to be involved in transactivator-mediated derepression of PAI-2 transcription in macrophage-like cells, as exemplified by the U937 cell line, c-Jun may be functional in other cell types. These data suggest a model for the transcriptional control of the human PAI-2 gene and further our understanding of the molecular basis for its tissue-specific expression.

The transcription factor C/EBP-ß mediates constitutive and LPS-inducible transcription of murine SerpinB2.

SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is highly induced in macrophages in response to inflammatory stimuli and is linked to the modulation of innate immunity, macrophage survival, and inhibition of plasminogen activators. Lipopolysaccharide (LPS), a potent bacterial endotoxin, can induce SerpinB2 expression via the toll-like receptor 4 (TLR4) by ∼1000-fold over a period of 24 hrs in murine macrophages. To map the LPS-regulated SerpinB2 promoter regions, we transfected reporter constructs driven by the ∼5 kb 5'-flanking region of the murine SerpinB2 gene and several deletion mutants into murine macrophages. In addition, we compared the DNA sequence of the murine 5' flanking sequence with the sequence of the human gene for homologous functional regulatory elements and identified several regulatory cis-acting elements in the human SERPINB2 promoter conserved in the mouse. Mutation analyses revealed that a CCAAT enhancer binding (C/EBP) element, a cyclic AMP response element (CRE) and two activator protein 1 (AP-1) response elements in the murine SerpinB2 proximal promoter are essential for optimal LPS-inducibility. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated that LPS induces the formation of C/EBP-ß containing complexes with the SerpinB2 promoter. Importantly, both constitutive and LPS-induced SerpinB2 expression was severely abrogated in C/EBP-ß-null mouse embryonic fibroblasts (MEFs) and primary C/EBP-ß-deficient peritoneal macrophages. Together, these data provide new insight into C/EBP-ß-dependent regulation of inflammation-associated SerpinB2 expression.

Investigation of the human stem cell factor KIT ligand gene, KITLG, in women with 46,XX spontaneous premature ovarian failure.

OBJECTIVE: To investigate mutations in the human KIT ligand gene (KITLG) gene as a mechanism of 46,XX spontaneous premature ovarian failure. The human KIT ligand gene, known also as human stem cell factor, is the ligand of the c-kit transmembrane tyrosine kinase receptor (KIT). This ligand-receptor interaction is known to play important roles in mouse germ cell migration and proliferation. DESIGN: Cross-sectional study. SETTING: Clinical research center. PATIENT(S): Forty women with 46,XX spontaneous premature ovarian failure. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Single-stranded conformational polymorphism analysis and DNA sequencing. RESULT(S): We found one nucleotide change of the KITLG coding region (811G-->T) that led to an alteration of the amino acid composition of the KITLG protein in one Caucasian patient (Asp210Tyr). However, we found the same alteration in two normal control Caucasian samples. Three nucleotide substitutions were found in the noncoding exon of KITLG (exon 10). We also identified two intronic polymorphisms. Thus, we did not identify a single significant mutation in the coding region of the KITLG gene in any of 40 patients (upper 95% confidence limit is 7.2%). CONCLUSION(S): Mutations in the coding regions of the KITLG gene appear not to be a common cause of 46,XX spontaneous premature ovarian failure in North American women.