Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant-associated biofilm infection and S. aureus bacteremia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease-dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared to the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison to the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease-mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.

Hormones that interact with seven‐ transmembrane spanning receptors, generally con‐sidered to be involved in acute signaling functions, also induce longer term effects on gene expression and cell growth. These genetic and proliferative effects can be induced by activation of receptors that signal through heterotrimeric GTP‐binding proteins (G‐proteins) of the Gq family, pertussis toxin‐sensitive Gi/Go proteins, Gs, or G12/G13. Numerous growth‐promoting G protein‐coupled receptors activate the low molecular weight G‐protein Ras and stimulate mitogen‐activated protein kinase. Recent data suggest that c‐Jun NH2‐terminal kinase is also activated, possibly through interaction with low molecular weight G‐proteins of the Rho family. Because G protein‐coupled receptors lack intrinsic tyrosine kinase activity, the mechanisms by which heterotrimeric G‐proteins couple to these kinase cascades remain to be elucidated. By analogy to growth factor receptors, G protein‐coupled receptors may access these kinase cascades through binding of adapter proteins or recruitment of cytosolic tyrosine kinases. It is likely that interactions between multiple signaling pathways are required for G protein‐coupled receptors to propagate signals to the nucleus.—Post, G. R., Brown, J. H. G protein‐coupled receptors and signaling pathways regulating growth responses. FASEBJ. 10, 741‐749 (1996)

In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac-specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene.

The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.

Objective-Telomerase serves as a critical regulator of tissue renewal. Although telomerase activity is inducible in response to various environmental cues, it remains unknown whether telomerase is activated during the inflammatory remodeling underlying atherosclerosis formation. To address this question, we investigated in the present study the regulation of telomerase in macrophages and during atherosclerosis development in low-density lipoprotein receptor-deficient mice. Methods and Results-We demonstrate that inflammatory stimuli activate telomerase in macrophages by inducing the expression of the catalytic subunit telomerase reverse transcriptase (TERT). Reporter and chromatin immunoprecipitation assays identified a previously unrecognized nuclear factor-B (NF-B) response element in the TERT promoter, to which NF-B is recruited during inflammation.

A novel clonal cell line derived from a human glioma (HOG) was found to express some oligodendrocyte-specific proteins including a 15-kDa form of myelin basic protein (MBP) and high 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Expression of the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide) was low. HOG cells did not express the characteristic astrocyte markers glial fibrillary acidic protein (GFAP) or significant glutamine synthetase (GS) activity. After initial plating, HOG cells were flat and epitheloid and thus showed a limited oligodendrocyte-like morphology. However, after cells became more confluent, some cells were phase-bright and elaborated short processes. Receptor types expressed by HOG cells included A2-adenosine, prostaglandin E1 (PGE1), and beta 2-adrenergic receptors (beta-ARs) linked to stimulation of adenylate cyclase, and muscarinic cholinergic and H1-histamine coupled to phosphatidyinositol turnover (Post and Dawson, 1991). HOG cells should therefore provide a useful model for studying the extracellular regulation and phosphorylation of oligodendrocyte-specific proteins.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G proteincoupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2', diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxininsensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that Ga12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.