Cyclic AMP acts through Rap1 and JNK signaling to increase expression of cutaneous smooth muscle alpha2C-adrenoceptors.

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of alpha(2C)-ARs from the transGolgi to the cell surface. The expression of alpha(2C)-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 muM), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 microM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH(2)-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 microM). Rap1CA increased the activity of an alpha(2C)-AR promoter-reporter construct, which was inhibited by SP600125 (3 microM) or by the mutation of an AP-1 binding site in the alpha(2C)-AR promoter. Furthermore, forskolin (10 microM) or CMC (100 microM) increased the expression of the alpha(2C)-AR protein, and these effects were inhibited by SP600125 (3 microM). Therefore, cyclic AMP increases the expression of alpha(2C)-ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the alpha(2C)-AR gene. By increasing the expression of cold-responsive alpha(2C)-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.

Estrogen increases smooth muscle expression of alpha2C-adrenoceptors and cold-induced constriction of cutaneous arteries.

Raynaud's phenomenon, which is characterized by intense cold-induced constriction of cutaneous arteries, is more common in women compared with men. Cold-induced constriction is mediated in part by enhanced activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) located on vascular smooth muscle cells (VSMs). Experiments were therefore performed to determine whether 17beta-estradiol regulates alpha(2C)-AR expression and function in cutaneous VSMs. 17beta-Estradiol (0.01-10 nmol/l) increased expression of the alpha(2C)-AR protein and the activity of the alpha(2C)-AR gene promoter in human cultured dermal VSMs, which was assessed following transient transfection of the cells with a promoter-reporter construct. The effect of 17beta-estradiol was associated with increased accumulation of cAMP and activation of the cAMP-responsive Rap2 GTP-binding protein. Transient transfection of VSMs with a dominant-negative mutant of Rap2 inhibited the 17beta-estradiol-induced activation of the alpha(2C)-AR gene promoter, whereas a constitutively active mutant of Rap2 increased alpha(2C)-AR promoter activity. The effects of 17beta-estradiol were inhibited by the estrogen receptor (ER) antagonist, ICI-182780 (1 micromol/l), and were mimicked by a cell-impermeable form of the hormone (estrogen:BSA) or by the selective ER-alpha receptor agonist 4,4',4'''-(4-propyl-[(1)H]-pyrazole-1,3,5-triyl)tris-phenol (PPT; 10 nmol/l) or the selective ER-beta receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nmol/l). Therefore, 17beta-estradiol increased expression of alpha(2C)-ARs by interacting with cell surface receptors to cause a cAMP/Rap2-dependent increase in alpha(2C)-AR transcription. In mouse tail arteries, 17beta-estradiol (10 nmol/l) increased alpha(2C)-AR expression and selectively increased the cold-induced amplification of alpha(2)-AR constriction, which is mediated by alpha(2C)-ARs. An estrogen-dependent increase in expression of cold-sensitive alpha(2C)-ARs may contribute to the increased activity of cold-induced vasoconstriction under estrogen-replete conditions.

Cooling evokes redistribution of alpha2C-adrenoceptors from Golgi to plasma membrane in transfected human embryonic kidney 293 cells.

Cold-induced vasoconstriction in cutaneous blood vessels is mediated by increased constrictor activity of vascular alpha2-adrenoceptors (alpha2-ARs). In mouse cutaneous arteries, alpha2-AR constriction at 37 degrees C is mediated by alpha2A-ARs, whereas after cold exposure (28 degrees C), alpha2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of alpha2-AR responsiveness. The goals of the present study were to develop a cell model of cutaneous thermoregulation and to determine the mechanisms underlying the thermosensitivity of alpha2C-ARs. Human embryonic kidney 293 cells were transiently transfected with the mouse alpha2A- or alpha2C-AR. In cells expressing alpha2A-ARs, UK-14,304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine), an alpha2-AR agonist, inhibited (10 pM) and stimulated (1-10 nM) the accumulation of cAMP evoked by forskolin. Similar responses were obtained at 37 degrees C and 28 degrees C. In contrast, in cells expressing alpha2C-ARs, UK-14,304 did not affect forskolin-stimulated cAMP accumulation at 37 degrees C but did cause a concentration-dependent inhibitory effect at 28 degrees C. Subcellular fractionation revealed that at 37 degrees C alpha2C-ARs were localized predominantly to Golgi compartments, whereas alpha2A-ARs localized predominantly to the plasma membrane. After cooling (28 degrees C), alpha2C-ARs relocated from Golgi compartments to the plasma membrane, whereas the alpha2A-AR remained at the plasma membrane. Immunofluorescence microscopy confirmed that, at 37 degrees C, alpha2A-ARs were localized to the cell surface, whereas alpha2C-ARs colocalized with a trans-Golgi marker. Cooling did not affect localization of alpha2A-ARs, but shifted alpha2C-ARs to the cell surface. Moderate cooling, therefore, caused a selective redistribution of alpha2C-ARs from the Golgi compartments to the cell surface, allowing the rescue of the alpha2C-adrenergic functional response. This mechanism may explain the role of alpha2-ARs in thermoregulation of the cutaneous circulation.

Redox regulation of vascular smooth muscle cell differentiation.

Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (alpha-actin, calponin, and SM1 and SM2 myosin), but not beta-actin. ROS activity, determined using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD; MnTMPyP, 25 micromol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC) decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L). None of the treatments altered beta-actin expression. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2). Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 micromol/L) reduced expression of differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38 MAPK-dependent pathway.

The small GTPases Ras, Rac, and Cdc42 transcriptionally regulate expression of human fibroblast growth factor 1.

Four distinct promoters (1A, 1B, 1C, and 1D) of fibroblast growth factor 1 (FGF1), spaced up to 70 kilobase pairs apart, direct the expression of alternatively spliced transcript variants (FGF1.A, -1. B, -1.C, and -1.D) that encode FGF1. These FGF1 transcripts can be detected in cultured cells as well as in normal and diseased tissues. These transcripts are differentially regulated in a cell-specific manner. To further delineate the biological function of multiple promoter usage by a single gene, we investigated the transcriptional regulation of these promoters by defined signaling pathways associated with cell proliferation and cell survival. Here we show a specific association of two of the FGF1 promoters, 1C and 1D, with signaling cascades of the Ras superfamily of GTPases. A serum-response element, comprised of the Ets and CArG motifs, present in promoter 1D was shown to be the target of distinct signaling cascades; the Ets motif target of Ras, Rac1, and Cdc42 regulation; and the CArG motif target of de novo protein synthesis-independent cascade. Ras and Rac1 also activated the FGF2 promoter. Further, the transcription factor Ets2 synergistically activated FGF1 gene, but not FGF2, in a Ras- and Rac1-dependent signaling pathway. In support of these conclusions high levels of intracellular FGF1 were detected in cells undergoing cytokinesis. Altogether, our results suggest that FGF1 may play a fundamental role in cell division, spreading, and migration, in addition to cell proliferation.

Silent alpha(2C)-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries.

Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle alpha(2)-adrenergic receptors (alpha(2)-ARs). Experiments were performed to determine the role of alpha(2)-AR subtypes (alpha(2A)-, alpha(2B)-, alpha(2C)-ARs) in this response. Stimulation of alpha(1)-ARs by phenylephrine or alpha(2)-ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to alpha(2)-AR activation but less responsive to activation of alpha(1)-ARs. Cold augmented constriction to alpha(2)-AR activation in distal arteries but did not affect the response to alpha(1)-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of alpha(2A)- and alpha(2C)-ARs in tail arteries: expression of alpha(2C)-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the alpha(2A)-AR increased in distal arteries. At 37 degrees C, alpha(2)-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of alpha(2A)-ARs (BRL-44408) but not by selective inhibition of alpha(2B)-ARs (ARC-239) or alpha(2C)-ARs (MK-912). In contrast, during cold exposure (28 degrees C), the augmented response to UK-14,304 was inhibited by the alpha(2C)-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of alpha(2)-ARs is mediated by alpha(2C)-ARs that are normally silent in these cutaneous arteries. Blockade of alpha(2C)-ARs may prove an effective treatment for Raynaud's Phenomenon.

Regulation of a promoter of the fibroblast growth factor 1 gene in prostate and breast cancer cells.

FGF-1 mRNA is expressed in the prostate cancer cell lines LNCaP and PC-3 and in the breast carcinoma cell line MDA-MB-231. Levels of FGF-1 mRNA have been shown to be up-regulated by serum, phorbol esters, and combinations of growth factors. It was shown that the major FGF-1 mRNA species expressed following serum stimulation in MDA-MB-231 cells is FGF-1.C. To better understand the potential role of FGF-1 in human prostate and breast cancer, we began an analysis of the cis- and trans-acting elements of one of its promoters required for the serum, PMA, and androgen regulation in breast and prostate cancer cell lines. We show that FGF-1.C steady-state mRNA levels are increased following serum or PMA stimulation of PC-3 cells. Further, we determine the FGF-1.C transcription start site in PC-3 cells. By sequence analysis, we show that consensus AP1, AP2, and Sp1 sites and ARE- and CRE-near consensus elements are present in the immediate 5' region of the FGF-1.C transcription start site. Gel-shift assays show that oligonucleotides containing FGF-1.C AP1, AP2, or Spl sequences form specific DNA-protein complexes with nuclear extracts from PC-3 cells. To determine if these or other cis-acting sequences are responsible for the serum, androgen, or growth factor regulation of FGF-1 expression, fragments of the FGF-1.C promoter region were cloned upstream of the luciferase reporter gene. We show that FGF-1 synergizes with androgen to enhance FGF-1.C transcription in LNCaP cells. We further show that the DNA fragment containing sequence up to 1614 nucleotides upstream of the FGF-1.C transcription start site is sufficient for stimulating promoter activity following serum treatment of MDA-MB-231 cells. Thus, FGF-1.C promoter contains sequences that are important for androgen or serum stimulation in prostate and breast cancer cells.

Differential regulation of human fibroblast growth factor 1 transcripts provides a distinct mechanism of cell-specific growth factor expression.

Four variants of fibroblast growth factor 1 (FGF-1) mRNA, FGF-1.A, -1.B, -1.C, and -1.D, originate from four discrete promoters of the gene. Each promoter is coupled with its 5'-untranslated exon. These four promoters are separated by as much as 70 kbp in the FGF-1 locus. The present study indicates that expression of these transcripts in different cell types is regulated by distinct mechanisms. FGF-1.C mRNA requires de novo protein synthesis and de novo transcription for expression and processing, and this mRNA increases acutely in response to TGF-beta and serum stimulation. The serum-induced FGF-1.C correlates with a marked increase in protein level. In addition, FGF-1.C mRNA also increases significantly (more than 100-fold) in response to phorbol 12-myristate 13-acetate. FGF-1.D mRNA is uniquely superinduced by serum in the presence of cycloheximide and displays delayed early kinetics, suggesting that this mRNA does not require de novo protein synthesis for expression. In sharp contrast to the FGF-1.C and -1.D mRNAs, FGF-1.B mRNA levels do not increase in response to serum or phorbol 12-myristate 13-acetate and are in fact slightly down-regulated. Furthermore, FGF-1.B mRNA is stable and appears to have a long half-life (> 12 h). Thus, the unique cell-specific regulation of these FGF-1 transcripts and subsequent protein synthesis indicate that each transcript may have a distinct role in development, normal cellular processes, and, upon aberrant regulation, disease. In support of these conclusions, multiple FGF-1 transcripts in normal, fetal, and diseased tissues, containing mixed cell types, were detected. Our results suggest that FGF-1 transcripts FGF-1.C and -1.D arising from promoters 1C and 1D are specific and are potential markers for proliferation of certain cells, whereas transcripts FGF-1.A and -1.B arising from promoters 1A and 1B are specific for maintenance and survival of cells, particularly cardiac and neuronal cells. Together, these data provide evidence for a biological function for multiple promoter usage of a single gene. The discrete mechanisms for expression of the FGF-1 gene further underscore the biological significance of this growth factor.

A recombinant PCR approach requiring only three non-chimeric primers to generate a minigene of interest.

The recombinant PCR allows construction of chimeric molecules. Here we describe this approach utilizing non-chimeric primers. Unlike previous recombinant PCR methods, this approach eliminates the need of multiple sets of primers and multiple rounds of PCR making it an economical and expeditious alternative. We have used this approach to generate an FGF-1 minigene.

Functional characterization of the brain-specific FGF-1 promoter, FGF-1.B.

Expression of alternatively spliced human FGF-1 (or aFGF) transcripts is regulated in a tissue-specific manner via multiple promoters. To identify the cis-regulatory elements in the brain-specific FGF-1.B promoter, we constructed a series of promoter deletions fused to the luciferase reporter gene and transfected into an FGF-1.B positive glioblastoma cell line, U1240MG, and a 1.B negative cell line, U1242MG. Results of transient transfections indicate three elements that are involved in the positive regulation of FGF-1.B expression. The core promoter is located in a 40-base pair region (between -92 and -49), and two regulatory regions (RR-1 and RR-2) are located within the 540-base pair region 5' to the major transcription start site (defined as +1). Electrophoretic mobility shift assays and footprinting analysis have identified sequence-specific binding sites in RR-1 and RR-2. Mutants of RR-2 abolished binding to nuclear proteins and showed diminished luciferase reporter activity. The effects seen are specific for the U1240MG cell line, supporting a role for RR-2 in the tissue-specific regulation of FGF-1.B. Southwestern analysis using an oligonucleotide probe derived from RR-2 (nucleotides -489 to -467) further identified a 37-kDa protein that is present in nuclear extracts from U1240MG and brain but not from U1242MG.

Human fibroblast growth factor 1 gene expression in vascular smooth muscle cells is modulated via an alternate promoter in response to serum and phorbol ester.

We have previously isolated the human FGF-1 gene in order to elucidate the molecular basis of its gene expression. The gene spans over 100 kbp and encodes multiple transcripts expressed in a tissue- and cell-specific manner. Two variants of FGF-1 mRNA (designated FGF-1.A and 1.B), which differ in their 5' untranslated region, were identified in our laboratory. Recently, two novel variants of FGF-1 mRNA (designated FGF-1.C and 1.D) have been isolated. In this study we used RNase protection assays to demonstrate expression of FGF-1.D mRNA in human fibroblasts and vascular smooth muscle cells and to show that promoter 1D has multiple transcription start sites. A single-strand nuclease-sensitive region has also been identified in the promoter 1D region that may have implications in chromatin conformation and transcriptional regulation of this promoter. Using Northern blot hybridization analyses, a previous study demonstrated a significant increase of FGF-1 mRNA levels in cultured saphenous vein smooth muscle cells in response to serum and phorbol ester. Here we confirm these results by RNase protection analysis and show that FGF-1.C mRNA is significantly increased in response to these stimuli. RNase protection assays indicate that promoter 1C has one major start site. The phorbol ester effect suggests that a protein kinase C-dependent signalling pathway may be involved in this phenomenon. Our results point to a dual promoter usage of the FGF-1 gene in vascular smooth muscle cells. Thus, normal growing cells primarily utilize promoter 1D. In contrast, quiescent cells, when exposed to serum or phorbol ester, utilize a different FGF-1 promoter, namely promoter 1C. Overall, these phenomena suggest mechanisms for increased production of FGF-1 that may play a role in inflammatory settings, wound healing, tissue repair, and neovascularization events and processes via autocrine and paracrine mechanisms. Our findings suggest that different FGF-1 promoters may respond to different physiological conditions and stimuli, in reference to the cell type or tissue milieu, resulting in ultimate production of the FGF-1 protein.

Cloning of two novel forms of human acidic fibroblast growth factor (aFGF) mRNA.

We have previously isolated two different aFGF cDNA clones from kidney and brain. The two corresponding mRNA, designated aFGF 1.A and 1.B, are the predominant species in kidney and brain, respectively. During the characterization of aFGF mRNA in glioblastoma cells, we demonstrated that aFGF mRNA in U1242MG and D65MG glioblastoma cells contain 5'-untranslated sequences different from those of 1.A and 1.B. Through a strategy combining chromosome walking, identification and sequencing of evolutionarily conserved DNA regions, and a reverse transcription and polymerase chain reaction (RT-PCR)-based assay for RNA expression, we have isolated two novel aFGF cDNA clones. The cDNA clone representing aFGF mRNA 1.C was isolated from U1242MG cells; another aFGF cDNA, designated 1.D, was isolated from D65MG cells. Promoter 1C has extensive sequence homology to the hamster aFGF gene promoter that was shown to respond to testosterone stimulation by chloramphenicol acetyltransferase reporter gene assays. Using RT-PCR, we showed that normal, benign and cancerous prostate tissues do not express aFGF 1.C mRNA. In contrast, a prostate carcinoma cell line (PC-3) expresses 1.C mRNA. RT-PCR using 1.D-specific primers showed that kidney, brain and prostate do not express 1.D mRNA even though kidney and brain are the most abundant source for aFGF protein. RNase protection analysis further showed that 1.D mRNA is the predominant aFGF transcript in D65MG glioblastoma cells and in NFF-6 neonatal foreskin fibroblast cells. The genomic DNA corresponding to these two cDNA clones and the 5'-flanking regions were also isolated and their sequences determined. These DNA clones will provide important reagents for studying the regulatory elements of aFGF gene expression.

Gene structure and differential expression of acidic fibroblast growth factor mRNA: identification and distribution of four different transcripts.

We have isolated four cDNA clones coding for human acidic fibroblast growth factor (aFGF) containing alternative 5' untranslated exons. Using RNAase protection analyses, we demonstrated the presence of at least four upstream, untranslated exons that are alternatively spliced to the first protein-coding exon. We designate these four untranslated exons, -1A, -1B, -1C and -1D. Splicing of these exons to the first coding exon will generate mRNA 1.A, 1.B, 1.C and 1.D respectively. Expression of these transcripts is regulated in a tissue-specific manner, as the major aFGF transcript in human brain frontal cortex differs from that in kidney. Furthermore, the pattern of aFGF transcripts in several glioblastoma cell lines tested is different from that in normal brain tissue. We isolated nine overlapping genomic clones containing these four upstream, untranslated exons. These four exons were localized on these clones by Southern hybridization and nucleotide sequence analysis. The overlapping clones are shown to be contiguous with our previously isolated genomic clones that contain the three aFGF-coding exons. The sizes of the four introns are 82.9, 71.1, 29.3 and 6.9 kbp. The transcriptional start sites of the two most upstream exons (-1A and -1B) have been mapped using RNAase protection and primer extension analyses. The sequences upstream of the start sites for aFGF 1.B mRNA do not contain a consensus TATA box. In contrast, the canonical CCAAT and TATA sequences are located at the proper distances from the transcription start site of aFGF 1.A mRNA.