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Gqα family members (Gqα, G11α, G14α, and G15/16α) stimulate phospholipase Cβ (PLCβ) and inositol lipid signaling but differ markedly in amino acid sequence and tissue distribution predicting unappreciated functional diversity. To examine functional differences, we compared the signaling properties of Gqα, G14α, and G15α and their cellular responses in vascular smooth muscle cells (VSMC). Constitutively active forms of Gqα, G14α, or G15α elicit markedly different responses when introduced to VSMC. Whereas each Gα stimulated PLCβ to similar extents when expressed at equal protein levels, Gqα and G14α but not G15α initiated profound cell death within 48 h. This response was the result of activation of apoptotic pathways, because Gqα and G14α, but not G15α, stimulated caspase-3 activation and did not alter phospho-Akt, a regulator of cell survival pathways. Gqα and G14α stimulate nuclear factor of activated T cell (NFAT) activation in VSMC, but Gα-induced cell death seems independent of PKC, InsP3/Ca2+, and NFAT, in that pharmacological inhibitors of these pathways did not block cell death. Gene expression analysis indicates that Gqα, G14α, and G15α each elicit markedly different profiles of altered gene sets in VSMC after 24 h. Whereas all three Gα stimulated changes (≥2-fold) in 50 shared mRNA, Gqα and G14α (but not G15α) stimulated changes in 221 shared mRNA, many of which are reported to be pro-apoptotic and/or involved with TNF-α signaling. We were surprised to find that each Gα also stimulated changes in nonoverlapping Gα-specific gene sets. These findings demonstrate that Gqα family members activate both overlapping and distinct signaling pathways and are more functionally diverse than previously thought.

Gqα family members (Gqα, G11α, G14α, and G15/16α) stimulate phospholipase Cβ (PLCβ) and inositol lipid signaling but differ markedly in amino acid sequence and tissue distribution predicting unappreciated functional diversity. To examine functional differences, we compared the signaling properties of Gqα, G14α, and G15α and their cellular responses in vascular smooth muscle cells (VSMC). Constitutively active forms of Gqα, G14α, or G15α elicit markedly different responses when introduced to VSMC. Whereas each Gα stimulated PLCβ to similar extents when expressed at equal protein levels, Gqα and G14α but not G15α initiated profound cell death within 48 h. This response was the result of activation of apoptotic pathways, because Gqα and G14α, but not G15α, stimulated caspase-3 activation and did not alter phospho-Akt, a regulator of cell survival pathways. Gqα and G14α stimulate nuclear factor of activated T cell (NFAT) activation in VSMC, but Gα-induced cell death seems independent of PKC, InsP3/Ca2+, and NFAT, in that pharmacological inhibitors of these pathways did not block cell death. Gene expression analysis indicates that Gqα, G14α, and G15α each elicit markedly different profiles of altered gene sets in VSMC after 24 h. Whereas all three Gα stimulated changes (≥2-fold) in 50 shared mRNA, Gqα and G14α (but not G15α) stimulated changes in 221 shared mRNA, many of which are reported to be pro-apoptotic and/or involved with TNF-α signaling. We were surprised to find that each Gα also stimulated changes in nonoverlapping Gα-specific gene sets. These findings demonstrate that Gqα family members activate both overlapping and distinct signaling pathways and are more functionally diverse than previously thought.

The prostaglandin synthase cyclooxygenase-2 (COX-2) is produced by an immediate early response gene induced in most cells by a variety of stimuli. Several studies have shown that the immunosuppressant cyclosporin (CsA) interferes with prostanoid metabolism, but the mechanisms are unclear. Here we examine the effect of CsA on COX-2 mRNA induction in cultured rat vascular smooth muscle cells (VSMC) that natively express the nuclear factor of activated T-cells, a known mediator of CsA-sensitive transcription. CsA significantly suppresses strong COX-2 mRNA induction caused by the Ca2+-mobilizing mitogens UTP, angiotensin II, and platelet-derived growth factor-BB, and the synergistic induction caused by costimulation with ionomycin and a phorbol ester. Forskolin and interleukin-1βare substantially weaker COX-2 mRNA inducers, and CsA does not inhibit their effect. CsA strongly inhibits UTP-, angiotensin II-, and platelet-derived growth factor-BB-stimulated COX-2 gene transcription as measured by nuclear run-on or promoter-reporter studies, but has no effect on mRNA induction caused by post-transcriptional stabilization of a distal COX-2 mRNA 3′-untranslated region regulatory element. These data show that CsA selectively inhibits mitogen-induced COX-2 gene expression by a transcriptional mechanism that may involve the nuclear factor of activated T-cells.

Activation of Gαq-coupled P2Y nucleotide receptors strongly (>100-fold) induces the rat vascular smooth muscle cell cyclooxygenase-2 (COX-2) mRNA, yet transcription is induced only ∼3-fold over 1 h. Intact cell decay analysis of tetracycline-suppressible luciferase chimera mRNAs shows that regulated stabilization of the intrinsically unstable mRNA contributes to this response. Deletion mapping of the 2468-base COX-2 mRNA 3′-untranslated region (UTR) shows that a distal, 130-base AU-rich region functions as a cis-acting regulated stabilization response element, which under basal conditions serves as the dominant instability determinant for the 3′-UTR. Regulation of this response is through the p42/44 MAP kinases, whereas the p38 MAP kinases are not involved. The stabilization response element binds avidly and specifically to a prominent nuclear-enriched ∼90-kDa factor and several less abundantly labeled mRNA binding proteins that are unaffected by P2Y receptor signaling. Although other instability determinants are located throughout the rat COX-2 mRNA 3′-UTR, mitogen signaling only interferes with rapid decay mediated by its most distal 130 bases. A complex of nuclear factors that bind this mRNA region specifically may include candidate targets for regulatory modulation. These observations support the general notion that the rapid induction of immediate-early gene expression through mitogenic receptors involves simultaneous activation of transcriptional and post-transcriptional mechanisms.

Cell surface receptor activation generally leads to changes in mRNA abundance, which may involve regulatory targets in processes working at the post-transcriptional level. Many types of agonists down-regulate vascular smooth muscle angiotensin receptor (AT1-R) gene expression, but it is unclear which of these activate post-transcriptional mechanisms. To reconstitute faithfully the normal AT1-R mRNA regulatory environment, tetracycline-suppressible promoters drive highly accurate recombinant AT1-R mRNA mimics in vascular smooth muscle cells that co-express an endogenous AT1-R mRNA. Down-regulation of the latter occurs shortly after stimulating mitogenic receptors or by using forskolin, but only cAMP signaling reduces expression of the recombinant AT1-R mRNA. Transcription of the recombinant mRNA is unaffected by cAMP signaling. Deletions of the AT1-R mRNA 3′-untranslated region do not impair cAMP-mediated down-regulation. Both loss of function and gain of function mutants show the response is mediated by the 5′-untranslated region. These observations provide the first direct functional evidence for modulation of vascular AT1-R gene expression by a mechanism involving a protein kinase A-regulated post-transcriptional process.

The nuclear factor of activated T cells (NFAT) mediates a cyclosporin A (CsA)- and FK506-suppressible transcriptional program in lymphocytes after antigen-stimulated phospholipase C activation. Nonlymphoid cells also express NFAT isoforms, raising the possibility that these isoforms can be regulated by other extracellular stimuli. This study sought to determine whether histamine can trigger NFAT-mediated transcription in human umbilical vein endothelial cells (HUVEC), using a retrovirus-based luciferase reporter driven by a well characterized, NFAT-specific enhancer. Luciferase levels are induced up to 60-fold over basal levels after costimulation of HUVEC with Ca2+-mobilizing drugs and a phorbol ester, a response that is 20-fold greater than that observed when HUVEC are stimulated with either drug alone. These synergistic responses are inhibited in cells treated with CsA. CsA and FK506 also inhibit the luciferase response to histamine, indicating that histamine can induce NFAT-mediated transcription in HUVEC. To identify candidate genes in HUVEC that might be regulated by NFAT, the expression of several chemokine mRNAs was measured after histamine treatment. Of the mRNAs tested, only those encoding monocyte chemotactic protein-1 (∼2-fold over basal) and interleukin-8 (∼6-fold over basal) are induced by histamine; both of these responses are suppressed by CsA and FK506. The H1histamine receptor antagonist chlorpheniramine, but not the H2receptor antagonist ranitidine, blocks the effects of histamine in this preparation. These data provide the first evidence for a physiological inducer of NFAT-mediated transcription in endothelial cells and support the hypothesis that NFAT participates in H1histamine receptor-induced interleukin-8 gene expression.