Ultraviolet B (UVB) irradiation potently induces cytokines in the skin, including interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α). The mechanism for TNF-α induction in UVB-irradiated keratinocytes is not clear. In the current study, we explored the effects of UVB and cytokines, alone or in combination in human keratinocytes. Keratinocytes were sham- or UVB-irradiated with 30 mJ/cm2, and then incubated in the absence or presence of IFN-α2b, TNF-α or IL-1α. UVB and IL-1α treatment synergistically enhanced TNF-α secretion and mRNA levels in human keratinocytes, similar to the findings reported previously in human fibroblasts. Exogenous recombinant TNF-α up-regulates its own mRNA level. However, addition of IFN-α2b did not show any additive effect on TNF-α mRNA induction. To understand the regulation of TNF-α mRNA by UVB, with or without IL-1α, we examined the transcription rate and half-life of TNF-α mRNA. Treatment of keratinocytes with IL-1α or UVB alone increased TNF-α gene transcription 4–5-fold over sham treatment, and TNF-α gene transcription increased 11-fold in cells treated with UVB plus IL-1α over sham. UVB with IL-1α did not enhance the half-life of TNF-α mRNA over that seen with UVB alone. In conclusion, TNF-α expression in primary keratinocytes is up-regulated transcriptionally by UVB and IL-1α.

Upregulation of TNF-alpha is a key early response to ultraviolet B (UVB) by keratinocytes (KCs), and represents an important component of the inflammatory cascade in skin. UVB irradiation induces TNF-alpha expression in both KCs and dermal fibroblasts, with TNF-alpha mRNA induction seen as early as 1.5 h after UVB. We previously reported that the effects are wavelength-specific: TNF-alpha expression and secretion are induced by UVB (290-320 nm), but not by UVA (320-400 nm). Moreover, we found that IL-1alpha, a cytokine also present in irradiated skin, substantially and synergistically enhances the induction of TNF-alpha by UVB, and the induction of TNF-alpha by this combination of UVB with IL-1alpha is mediated through increased TNF-alpha gene transcription. We investigated the molecular mechanism for UVB-induction of the TNF-alpha gene with a series of TNF-alpha promoter constructs, ranging from 1.2 kbp (from -1179 to +1 with respect to the TNF-alpha transcription initiation site) down to 0.1 kbp (-109 to +1), each driving expression of a CAT reporter. Our results showed a persistent nine to tenfold increase of CAT activity in all TNF-alpha promoter/reporter constructs in response to UVB (30 mJ/cm(2)) exposure. These results indicate the presence of UVB-responsive cis-element(s) located between -109 and +1 of the TNF-alpha promoter, a region that contains a putative AP-1 site and a putative NFkB site. UVB-induction was abolished when the TNF-alpha promoter was mutated by one base pair at the AP-1 binding site. Cells treated with SP600125, an AP-1 inhibitor that inhibits JNK (c-Jun N-terminal kinase), also showed suppression of the 0.1 kbp TNF-alpha promoter/reporter construct. The authentic endogenous gene in untransfected cells was also blocked by the inhibitor. Electrophoretic Mobility Shift Assay indicated new complexes from UVB-treated nuclear extracts and anti-phospho-c-Jun, a regulatory component of the AP-1 transcription factor, creating a supershift indicating increased phosphorylation of c-Jun and hence higher AP-1 activity. Keratinocyte-derived TNF-alpha is a component of the early induction phase of the inflammatory cascade.

In most human follicular lymphomas, the chromosome translocation t(14;18) occurs within two breakpoint clustering regions on chromosome 18, the major one at the 3' untranslated region of the bc1-2 gene and the minor one at 3' of the gene. Analysis of a panel of follicular lymphoma DNAs using probes for the first exon of the bc1-2 gene indicates that DNA rearrangements may also occur 5' to the involved bcl-2 gene. In this case the IgH locus and the bc1-2 gene are found in the order 3' C.y Sy/1, JH 5'::5' bcl-2 3' (where C = constant, S = switch, and JH = joining segment of the heavy chain locus), suggesting that an inversion also occurred during the translocation process. The coding regions of the bc1-2 gene, however, are left intact in all cases of follicular lymphoma studied to date.More than 80% of human follicular lymphomas carry a t(14;18)(q32;q21) chromosome translocation (1, 2). This translocation directly involves the immunoglobulin heavy chain (IgH) locus and the bc1-2 (B-cell lymphoma/leukemia 2) gene (3,4). By comparing the structures of bc1-2 cDNA clones and genomic DNA clones, we have shown that the human bc1-2 gene consists of at least two exons separated by an intron of >50 kilobases (kb) of DNA (5). The first exon is transcribed into a 3.5-kb mRNA (5) (also see Fig. 1). This exon contains a splicing donor signal so that bc1-2 mRNA is spliced to the second exon to produce a 5.5-kb and an 8.5-kb mRNA (5) (also see Fig. 1). The 5.5-kb and 3.5-kb mRNA code for the bcl-2 a and , protein, respectively, which are identical except for the carboxyl-terminal portion (5). We have shown that '60% of the breakpoints of the t(14;18) translocation on chromosome 18 are tightly clustered in the 3' noncoding region of the bc1-2 gene (3-5) and -10% are clustered at a region 3' to the bc1-2 gene (3, 4) (also see Fig. 1). We have also shown that the association of the bc1-2 gene with the heavy chain locus results in high levels of bc1-2 expression (3). In all cases we have analyzed previously, the immunoglobulin heavy chain (IgH) locus, including the IgH enhancer, is 3' to the involved bcl-2 open reading frames (3-5), suggesting that the IgH locus is responsible for the bc1-2 activation in the follicular lymphomas. MATERIALS AND METHODSGel Electrophoresis and Southern Transfer. High molecular weight DNA was digested with restriction endonucleases and 5-,ug samples were fractionated on 0.7% agarose gels and transferred to nitrocellulose filters essentially as described by Southern (6).Construction of Genome 2 DNA Library. DNA extracted from the follicular lymphoma FL989 was partially digested with restriction enzyme Sau3AI and DNA fragments between 15 and 23 kb were collected. DNA inserts were ligated with XEMBL3A phage vector DNA cut with BamHI and packaged in vitro (7,8). Two recombinant phage clones, containing a fragment corresponding in size to the rearranged bcl-2 first exon sequences, were obtained by screening the library with the bcl-2 first exon probe pB16 (5).DNA Sequencing. Nucleotide sequences wer...

Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.Elastin is a resilient connective tissue protein present abundantly in tissues such as skin and lung, and in the large blood vessels of the cardiovascular system. Aortic elastin fibers, assembled extracellularly from cross-linked tropoelastin monomers and microfibrillar proteins, allow tissues of the cardiovascular system to undergo repeated stretching and recoil, a necessary property for maintaining physiologic pressure gradients. Production of elastin, like other connective tissue proteins, is developmentally regulated. The greatest elastogenic period occurs during late fetal and early neonatal periods (1-4), followed by a drop in elastin production and mRNA steady state levels in adulthood (5-7). However, in response to injury such as in the atherosclerotic lesion, elastin production is upregulated (8). Lesions are accompanied by a rise in a variety of growth factors, including IGF-I, 1 which is generally not expressed in the adult artery (8). Since there is a significant temporal correlation between the rise of IGF-I levels in blood plasma (4), the initial burst of developmental aortic elastogenesis (1-4), and the production of a connective tissue matrix accompanying the atherosclerotic plaque (8), elucidation of mechanisms whereby IGF-I increases elastogenesis is of great interest.Recently our laboratory has shown that the increase in elastin gene transcription elicited by IGF-I in aortic SMC is accompanied by a loss of DNA-protein binding to an inhibitor sequence in the proximal promoter (9). In situ footprint analysis of the deprotected region coupled with results from transient transfections performed with deletion constructs suggested that the specific sequence affected is Ϫ165 to Ϫ137 bp (EFE 5/6) of the promoter. Gel shift analysis of an oligomer representing this sequence with nuclear extract isolated from control SMC showed complex formation with Sp1...