Activation of the alpha-smooth muscle actin (alpha-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates alpha-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased alpha-SMA expression is controlled at the transcriptional level because the alpha-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the alpha-SMA gene was mimicked by overexpression of constitutively active Galphai2 (alphai2Q205L) but not Galpha16 (alpha-16Q212L). Blockade of hypoxia-induced alpha-SMA expression with pertussis toxin, a Galphai antagonist, confirmed a role for Galphai in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated alpha-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and alpha-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-beta1 blocked only 30% of the hypoxia-induced alpha-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of alpha-SMA, and this increase in alpha-SMA level occurs through Galphai- and JNK-dependent signaling pathways.

Much of the gene regulatory circuitry of phage centers on a complex region called the O R region. This ϳ100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a cloning vector that allowed us to clone fragments spanning the O R region. By using this vector, we carried out intensive mutagenesis of the P RM promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the ؊35 and ؊10 regions were randomized and cloned this pool into the vector, making a pool of P RM variant phage. About 10% of the P RM variants were able to lysogenize, suggesting that the regulatory circuitry is compatible with a wide range of P RM sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.Complex gene regulatory circuits can have a large number of interlocking components. This degree of interconnectivity raises two issues. First, how did these circuits evolve? Second, how can we understand the behavior of these existing circuits and predict their behavior in the face of small changes in parameters such as promoter strength? For these and other reasons, we have been analyzing the behavior of the regulatory circuitry of phage in the intact system. This system is probably the best-understood complex circuit (38, 39). Most, if not all, of the regulatory interactions have been identified, and most of these are well characterized at the mechanistic level. Previous analysis of this circuit generally has been carried out in uncoupled systems (such as the use of reporter genes and fusions with the lac promoter), an approach necessary to disentangle the causality of this system. With a circuit diagram in hand, it is now possible to return to the intact circuit and ask how particular changes affect the overall operation of the system.Many of the critical interactions in the circuit center on a complex regulatory region termed the O R region (Fig. 1C). This ϳ100-bp region is densely packed with cis-acting sites, including two promoters and three sites to which both the CI and Cro repressors can bind (38). In addition, the promoters and repressor-binding sites overlap extensively. CI and Cro regulate the expr...

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Novel selenazolidine prodrugs of selenocysteine are being developed as potential selenium delivery agents for cancer chemoprevention and other clinical uses. The 2-unsubstituted compound, selenazolidine-4(R)-carboxylic acid (L-SCA), and the 2-oxo- and 2-methyl analogues possessing D-stereochemistry (D-OSCA and D-MSCA, respectively) were synthesized and chemically characterized. L/D pairs, along with other organoselenium compounds and common inorganic forms, were studied in cultured V79 cells to understand their inherent toxicity and their ability to induce selenium-dependent glutathione peroxidase (GPx) activity, which indicates the provision of biologically available selenium. All of the selenazolidines were much less toxic to the cells than was sodium selenite (IC(50) approximately 17 microM) or the parent selenolamines, L- or D-selenocystine (IC(50) approximately 34 or 39 microM, respectively); OSCA was less toxic than MSCA. The stereoisomers of OSCA produced very different IC(50) values (L-OSCA, approximately 451 microM; D-OSCA, >3000 microM), while the IC(50) values derived for the stereoisomers of MSCA were of the same order of magnitude (L-MSCA, approximately 79 microM; D-MSCA, approximately 160 microM). Compounds possessing L-stereochemistry were at least as active with respect to GPx induction as was sodium selenite (2.2-fold increase at 15 microM). L-Selenocystine produced a 4.2-fold increase in GPx activity at 30 microM, while L-SCA produced a 5.9-fold increase, followed by L-OSCA (4.6-fold) and L-MSCA (2.1-fold), all at 100 microM. Compounds possessing D-stereochemistry showed minimal ability to induce GPx activity (D-selenocystine, 1.0-fold increase; D-OSCA, 1.4-fold increase; D-MSCA, 1.3-fold increase).

We have previously found that hypoxia stimulates proliferation of vascular fibroblasts through Galphai-mediated activation of ERK1/2. Here, we demonstrate that hypoxia also activates the atypical protein kinase Czeta (PKCzeta) isozyme and stimulates the expression of ERK1/2-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), which attenuates ERK1/2-mediated proliferative signals. Replication repressor activity is unique to PKCzeta because the blockade of classical and novel PKC isozymes does not affect fibroblast proliferation. PKCzeta is phosphorylated upon prolonged (24 h) exposure to hypoxia, whereas ERK1/2, the downstream kinases, are maximally activated in fibroblasts exposed to acute (10 min) hypoxia. However, PKCzeta blockade results in persistent ERK1/2 phosphorylation and marked increase in hypoxia-induced replication. Similarly prolonged ERK1/2 phosphorylation and increase in hypoxia-stimulated proliferation are also observed upon blockade of MKP-1 activation. Because of the parallel suppressive actions of PKCzeta and MKP-1 on ERK1/2 phosphorylation and proliferation, the role of PKCzeta in the regulation of MKP-1 expression was evaluated. PKCzeta attenuation reduces MKP-1 expression, whereas PKCzeta overexpression increases MKP-1 levels. In conclusion, our results indicate for the first time that hypoxia activates PKCzeta, which acts as a terminator of ERK1/2 activation through the regulation of downstream target, MKP-1 expression and thus serves to limit hypoxia-induced proliferation of fibroblasts.