Search

The tuberous sclerosis complex (TSC) is caused by mutation in either of 2 tumor suppressor genes, TSC-1(encodes hamartin) and TSC-2(encodes tuberin). In humans, deficiency in TSC1/2is associated with benign tumors in many organs, including renal angiomyolipoma (AML) but rarely renal cell carcinoma (RCC). In contrast, deficiency of TSCfunction in the Eker rat is associated with RCC. Here, we have investigated the activity of PI 3-K and the expression of PTEN, p53, tuberin, p-mTOR, and p-p70S6K in both Eker rat RCC and human renal AML. Compared to normal tissue, increased PI 3-K activity was detected in RCC of Eker rats but not in human AML tissue. In contrast, PTENwas highly expressed in AML but significantly reduced in the renal tumors of Eker rats. Phosphorylation on Ser2448of mTOR and Thr389of p70S6K were significantly increased in both RCC and AML compared to matching control tissue. Total tuberin was significantly decreased in AML while completely lost in RCC of Eker rats. Our data also show that while p53 protein expression is lost in rat RCC, it was highly elevated in AML. These novel data provide evidence that loss of TSC-2, PTEN, and p53as well as activation of PI 3-K and mTOR is associated with kidney cancer in the Eker rat, while sustained expression of TSC-2, PTEN, and p53may prevent progression of kidney cancer in TSC patients.

Macrophages have the potential to deliver therapeutic genes to many target tissues. Macrophage-specific synthetic promoters (SPs) generated by random ligation of myeloid/macrophage cis elements had activity up to 100-fold that of a native macrophage promoter in macrophage cell lines, but were minimally active in nonmyeloid cells. Mouse bone marrow cells (BMCs) transduced ex vivo with lentivectors expressing green fluorescent protein (GFP) driven either by an SP (SP-GFP) or a cytomegalovirus (CMV) promoter (CMV-GFP) were used for syngeneic transplantation of lethally irradiated mice. Blood leukocytes showed stable GFP expression for up to 15 months after transplantation. SP-GFP expression was selective for CD11b+ macrophages, whereas CMV-GFP expression was observed in erythrocytes, as well as in both CD11b+ and CD11b– leukocytes. Furthermore, SP-GFP expression was much stronger than CMV-GFP expression in CD11b+ macrophages. apoE–/– BMCs transduced with the lentiviral vector encoding human apoE were used to transplant apoE–/– mice. Macrophage expression of apoE from 10 to 26 weeks of age significantly reduced atherosclerotic lesions in recipient apoE–/– mice. Thus, the novel SPs, especially when combined with lentivectors, are useful for macrophage-specific delivery of therapeutic genes.

Induction of differentiation of HL-60 human myeloid cells profoundly affected expression of calreticulin, a Ca(2+)-binding endoplasmic reticulum chaperone. Induction with Me(2)SO or retinoic acid reduced levels of calreticulin protein by approximately 60% within 4 days. Pulse-chase studies indicated that labeled calreticulin decayed at similar rates in differentiated and undifferentiated cells (t(12) approximately 4.6 days), but the biosynthetic rate was <10% of control after 4 days. Differentiation also induced a rapid decline in calreticulin mRNA levels (90% reduction after 1 day) without a decrease in transcript stability (t(12) approximately 5 h). Nuclear run-on analysis demonstrated rapid down-regulation of gene transcription (21% of control at 2 h). Differentiation also greatly reduced the Ca(2+) content of the cells (25% of control), although residual Ca(2+) pools remained sensitive to thapsigargin, ionomycin, and inositol trisphosphate. Progressive decreases were also observed in levels of calnexin and ERp57, whereas BiP/GRP78 and protein disulfide isomerase were only modestly affected. Ultrastructural studies showed a substantial reduction in endoplasmic reticulum content of the cells. Thus, terminal differentiation of myeloid cells was associated with decreased endoplasmic reticulum content, selective reductions in molecular chaperones, and diminished intracellular Ca(2+) stores, perhaps reflecting an endoplasmic reticulum remodeling program as a prominent feature of granulocytic differentiation.

The p40phox protein, a regulatory component of the phagocyte NADPH oxidase, is preferentially expressed in cells of myeloid lineage. We investigated transcriptional regulation of thep40phox gene in HL-60 myeloid cells. Deletion analysis of approximately 6 kb of the 5′-flanking sequence of the gene demonstrated that the proximal 106 base pair of the promoter exhibited maximum reporter activity. This region contains 3 potential binding sites for PU.1, a myeloid-restricted member of theets family of transcription factors. Mutation or deletion of each PU.1 site decreased promoter activity, and the level of activity mediated by each site correlated with its binding avidity for PU.1, as determined by gel shift competition assays. Mutation of all 3 sites abolished promoter activity in myeloid cells. PU.1-dependent expression was also observed in the Raji B-cell line, whereas the moderate level of promoter reporter activity in the nonmyeloid HeLa cell line was independent of PU.1. Chromatin immunoprecipitation assay demonstrated occupation of the PU.1 sites by PU.1 in vivo in HL-60 cells. Cotransfection of the pGL3-p40-106 reporter construct with a dominant-negative PU.1 mutant dramatically reduced promoter activity, whereas the overexpression of PU.1 increased promoter activity. Promoter activity and transcript levels ofp40phox increased in HL-60 cells during dimethyl sulfoxide–induced differentiation toward the granulocyte phenotype, and this was associated with increased cellular levels of PU.1 protein. Our findings demonstrate that PU.1 binding at multiple sites is required for p40phox gene transcription in myeloid cells and that granulocytic differentiation is associated with the coordinated up-regulation of PU.1 andp40phox expression.

The p40phoxprotein, a regulatory component of the phagocyte NADPH oxidase, is preferentially expressed in cells of myeloid lineage. We investigated transcriptional regulation of thep40phoxgene in HL-60 myeloid cells. Deletion analysis of approximately 6 kb of the 5′-flanking sequence of the gene demonstrated that the proximal 106 base pair of the promoter exhibited maximum reporter activity. This region contains 3 potential binding sites for PU.1, a myeloid-restricted member of theetsfamily of transcription factors. Mutation or deletion of each PU.1 site decreased promoter activity, and the level of activity mediated by each site correlated with its binding avidity for PU.1, as determined by gel shift competition assays. Mutation of all 3 sites abolished promoter activity in myeloid cells. PU.1-dependent expression was also observed in the Raji B-cell line, whereas the moderate level of promoter reporter activity in the nonmyeloid HeLa cell line was independent of PU.1. Chromatin immunoprecipitation assay demonstrated occupation of the PU.1 sites by PU.1 in vivo in HL-60 cells. Cotransfection of the pGL3-p40-106 reporter construct with a dominant-negative PU.1 mutant dramatically reduced promoter activity, whereas the overexpression of PU.1 increased promoter activity. Promoter activity and transcript levels ofp40phoxincreased in HL-60 cells during dimethyl sulfoxide–induced differentiation toward the granulocyte phenotype, and this was associated with increased cellular levels of PU.1 protein. Our findings demonstrate that PU.1 binding at multiple sites is required for p40phoxgene transcription in myeloid cells and that granulocytic differentiation is associated with the coordinated up-regulation of PU.1 andp40phoxexpression.

We have investigated the myeloid-specific transcriptional regulation of p67phox, an essential component of phagocyte respiratory burst NADPH oxidase. Analysis was carried out on the p67phox5′-flanking region from –3669 to –4 (relative to ATG), including the first exon and intron and part of the second exon. The construct extending from –985 to –4 produced the highest luciferase activity in myeloid HL-60 cells but was not active in HeLa or Jurkat cells, indicating myeloid-specific expression. Four active elements were identified: Sp1/Sp3 at –694, PU.1 at –289, AP-1 at –210, and PU.1/HAF1 at –182, the latter three being in the first intron. These ciselements bound their cognate transacting factors both in vitroand in vivo. Mutation of the Sp1, PU.1, or PU.1/HAF1 site each decreased promoter activity by 35–50%. Mutations in all three sites reduced promoter activity by 90%. However, mutation of the AP-1 site alone nearly abolished promoter activity. The AP-1 site bound Jun and Fos proteins from HL-60 cell nuclear extract. Co-expression with Jun B in AP-1-deficient cells increased promoter activity by 3-fold. These data show that full p67phoxpromoter activity requires cooperation between myeloid-specific and nonmyeloid transcription factors, with AP-1 being the most critical for function.

Polymorphisms in CC chemokine receptor 5 (CCR5), the major coreceptor of human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV), have a major influence on HIV-1 transmission and disease progression. The effects of these polymorphisms may, in part, account for the differential pathogenesis of HIV-1 (immunosuppression) and SIV (natural resistance) in humans and non-human primates, respectively. Thus, understanding the genetic basis underlying species-specific responses to HIV-1 and SIV could reveal new anti-HIV-1 therapeutic strategies for humans. To this end, we comparedCCR5structure/evolution and regulation among humans, apes, Old World Monkeys, and New World Monkeys. The evolution of theCCR5 cis-regulatory region versusthe open reading frame as well as among different domains of the open reading frame differed from one another. CCR5 cis-regulatory region sequence variation in humans was substantially higher than anticipated. Based on this variation, CCR5haplotypes could be organized into seven evolutionarily distinct human haplogroups (HH) that we designated HHA, -B, -C, -D, -E, -F, and -G. HHA haplotypes were defined as ancestral to all other haplotypes by comparison to theCCR5haplotypes of non-human primates. Different human and non-human primate CCR5haplotypes were associated with differential transcriptional regulation, and various polymorphisms resulted in modified DNA-nuclear protein interactions, including altered binding of members of the NF-κB family of transcription factors. We identified novel CCR5 untranslated mRNA sequences that were conserved in human and non-human primates. In some primates, mutations at exon-intron boundaries caused loss of expression of selected CCR5 mRNA isoforms or production of novel mRNA isoforms. Collectively, these findings suggest that the response to HIV-1 and SIV infection in primates may have been driven, in part, by evolution of the elements controlling CCR5 transcription and translation.

The myeloid-specific transcription factor PU.1 is essential for expression of p47phox, a component of the superoxide-forming phagocyte NADPH oxidase. The consensus PU.1 binding sequence (GAGGAA) is located on the non-coding strand from position −40 to −45 relative to the transcriptional start site of thep47phoxpromoter. A promoter construct extending to −46 was sufficient to drive tissue-specific expression of the luciferase reporter gene, but extension of the promoter from −46 to −48 resulted in a significant increase in reporter expression. Mutations of the nucleotides G at −46 and/or T at −47 reduced both reporter expression and PU.1 binding, whereas mutations at −48 had no effect. The PU.1 binding avidity of these sequences correlated closely with their capacity to dictate reporter gene transcription. In parallel studies on the functional PU.1 site in the promoter of CD18, mutations of nucleotides G and T at positions −76 and −77 (corresponding to −46 and −47, respectively, of the p47phoxpromoter) reduced PU.1 binding and nearly abolished the contribution of this element to promoter activity. We conclude that the immediate flanking nucleotides of the PU.1 consensus motif have significant effects on PU.1 binding avidity and activity and that this region is the dominantciselement regulating p47phoxexpression.

A monocyte chemotactic protein (MCP-1) is thought to play a major role in recruiting monocytes to the vascular endothelium where the adherence of monocytes is one of the earliest events in atherogenesis. We cloned MCP-1 cDNA from a ?gt 11 cDNA library constructed from human aortic endothelial mRNA to test whether MCP-1 expressed in arterial endothelium is identical to those from other sources. A ~ 670 bp MCP-1 cDNA clone was identified and showed the identical sequence with the ones from other cell lines. Northern blot analysis using this cloned MCP-1 cDNA as probe revealed two hybridizing bands of RNA at 0.68 and 0.77 kb in human aortic, human pulmonary arterial, and human umbilical vein endothelial cell cultures. Primer extension analysis showed that the difference in size (~ 90 bp) between the two transcripts is not due to a difference at the 5'-noncoding region. The amount of MCP-1 transcripts increased dramatically in aortic endothelial cells when stimulated with recombinant IL-1a (100 units/ml), IL-1ß (100 units/ml), or TNF-a (200 ng/ml). Northern blot and slot blot analysis of RNA isolated from both the endothelium and the underlying vessel wall of freshly removed human arteries and veins showed MCP-1 transcripts. This observation demonstrates for the first time that MCP-1 is expressed not only in atherosclerotic human arteries but also in symptom free arteries and veinsin vivo.

The ultrastructural features of peripheral blood monocyte margination, migration, and aortic intimal accumulation have been described in the normo- and mildly hypercholesterolemic baboon. Intimal monocyte-macrophage recruitment over fatty streaks and fibro-fatty plaques was enhanced by dietary cholesterol-fat supplementation, resulting in an 8-fold increase in monocyte-macrophages and macrophage-derived foam cells in the subendothelial space. Margination or attachment observed over both plaques and normal areas was not associated with morphologic evidence of endothelial injury. Migration through continuous aortic endothelium was principally between endothelial cells via junctions. Transitional sequences from the typical morphology of the blood monocyte to the lipid-containing macrophage or foam cell were discerned. The intimal accumulation of monocytes and macrophages reinforces our view of atherosclerosis as an inflammatory process, in which monocyte attachment is likely to reflect changes in the endothelial surface-membrane complex and surface charge, while migration to and accumulation in the SES may result from one or more chemoattractants originating in the intima or media.

This synopsis emphasizes the inappropriateness of a “single stimulus-single response” approach in understanding the response of the arterial wall to injury and repair. The outcome of any injurious stimulus is a series of interactive cascades among the endogenous and exogenous cellular and non-cellular components of the arterial wall, and the cellular and non-cellular elements of the blood. Both genetic and hemodynamic factors can further influence this response. The more prominent of the cellular and non-cellular components have been discussed. These include: 1) the vascular endothelium, its dynamic interaction with macromolecules and formed elements of the blood, its role in the transport of plasma proteins, its influence on the function of arterial smooth muscle cells and the recruitment of blood-born monocytes; 2) the arterial smooth muscle cell, its role in the vasomotor function of the artery wall, arterial repair and reconstruction, metabolism of lipids and the secretion of cytokines regulating monocyte recruitment; 3) the mononuclear phagocyte, its role in arterial debridement, metabolism of modified LDLs, a precursor of the cholesteryl ester-rich foam cell, and the secretion of neutral hydrolases, bioactive lipids and cytokines; 4) lymphocytes, as mediators of the inflammatory response and possible autoimmune reactions; 5) platelets, their roles in hemostasis, thrombosis, atherogenesis, and the repair process and 6) plasma LDLs, their oxidative modification by cells of the vessel wall and their roles in the injury process. The interactive processes among arterial and circulating components in both injury and repair is emphasized.

Monocyte chemoattractant protein 1 (MCP‐1) is a member of the chemokine superfamily of genes that induces chemotaxis of monocytes in inflammatory processes. The effects of interleukin‐1β (IL‐1β), tumor necrosis factor α (TNF‐α), interleukin‐6 (IL‐6), transforming growth factor β (TGF‐β), platelet‐derived growth factor (PDGF‐BB), parathyroid hormone (PTH), and 1,25(OH)2D3on MCP‐1 expression in human osteoblastic cells were compared. Inflammatory or proinflammatory cytokines stimulated the production of MCP‐1 in normal human osteoblastic cells as determined by RIA. The osteotrophic mediators PTH and 1,25(OH)2D3and PDGF‐BB had no effect on MCP‐1 expression. In further studies, the steady‐state mRNA and MCP‐1 protein levels in two human osteoblastic cell lines, MG‐63 and SaOS‐2, were examined. MCP‐1 expression at both the protein and mRNA levels was greatly increased by IL‐1β and TNF‐α. At the mRNA level, IL‐1β and TNF‐α strongly induced MCP‐1 expression; TGF‐β and IL‐6 induced MCP‐1 but to a lesser extent. No significant changes in MCP‐1 mRNA or MCP‐1 protein secretion were observed when cells were treated with PDGF‐BB, PTH, and 1,25(OH)2D3. When tested on preosteoclasts, MCP‐1 was shown to have no effect on the formation of multinucleated, tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclastic cells.

Expression of the phagocyte cytosolic protein p47 phox, a component of NADPH oxidase, is restricted mainly to myeloid cells. To study the cis-elements and trans-acting factors responsible for its gene expression, we have cloned and characterized the p47phoxpromoter. A predominant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon. To identify the gene promoter sequences, transient transfections of HL-60 human myeloid cells were performed with a series of 5′-deletionp47phox-luciferase reporter constructs that extended as far upstream as −3050 bp relative to the transcriptional start site. The −224 and −86 constructs had the strongestp47phoxpromoter activity, whereas the −46 construct showed a major reduction in activity and the −36 construct a complete loss of activity. DNase I footprint analysis identified a protected region from −37 to −53. This region containing a consensus PU.1 site bound specifically both PU.1 present in nuclear extracts from myeloid cells and PU.1 synthesized in vitro. Mutations of this site eliminated PU.1 binding and abolished the ability of thep47phoxpromoter to direct expression of the reporter gene. Thep47phoxpromoter was active in all myeloid cell lines tested (HL-60, THP-1, U937, PLB-985), but not in non-myeloid cells (HeLa, HEK293). Finally, PU.1 trans-activated thep47phox-luciferase constructs in HeLa cells. We conclude that, similar to certain other myeloid-specific genes,p47phoxpromoter activity in myeloid cells requires PU.1.