Selective expression of an endogenous inhibitor of FAK regulates proliferation and migration of vascular smooth muscle cells.

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [(3)H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.

Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization.

Smooth muscle cell (SMC) differentiation is regulated by a complex array of local environmental cues, but the intracellular signaling pathways and the transcription mechanisms that regulate this process are largely unknown. We and others have shown that serum response factor (SRF) contributes to SMC-specific gene transcription, and because the small GTPase RhoA has been shown to regulate SRF, the goal of the present study was to test the hypothesis that RhoA signaling is a critical mechanism for regulating SMC differentiation. Coexpression of constitutively active RhoA in rat aortic SMC cultures significantly increased the activity of the SMC-specific promoters, SM22 and SM alpha-actin, whereas coexpression of C3 transferase abolished the activity of these promoters. Inhibition of either stress fiber formation with the Rho kinase inhibitor Y-27632 (10 microm) or actin polymerization with latrunculin B (0.5 microm) significantly decreased the activity of SM22 and SM alpha-actin promoters. In contrast, increasing actin polymerization with jasplakinolide (0.5 microm) increased SM22 and SM alpha-actin promoter activity by 22-fold and 13-fold, respectively. The above interventions had little or no effect on the transcription of an SRF-dependent c-fos promoter or on a minimal thymidine kinase promoter that is not SRF-dependent. Taken together, the results of these studies indicate that in SMC, RhoA-dependent regulation of the actin cytoskeleton selectively regulates SMC differentiation marker gene expression by modulating SRF-dependent transcription. The results also suggest that RhoA signaling may serve as a convergence point for the multiple signaling pathways that regulate SMC differentiation.

Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex.

Previous studies have shown that multiple serum response factor (SRF)-binding CArG elements were required for smooth muscle cell (SMC)-specific regulation of smooth muscle (SM) alpha-actin expression. However, a critical question remains as to the mechanisms whereby a ubiquitously expressed transcription factor such as SRF might contribute to SMC-specific expression. The goal of the present study was to investigate the hypothesis that SMC-selective expression of SM alpha-actin is due at least in part to (1) unique CArG flanking sequences that distinguish the SM alpha-actin CArGs from other ubiquitously expressed CArG-dependent genes such as c-fos, (2) cooperative interactions between CArG elements, and (3) SRF-dependent binding of SMC-selective proteins to the CArG-containing regions of the promoter. Results demonstrated that specific sequences flanking CArG B were important for promoter activity in SMCs but not in bovine aortic endothelial cells. We also provided evidence indicating that the structural orientation between CArGs A and B was an important determinant of promoter function. Electrophoretic mobility shift assays and methylation interference footprinting demonstrated that a unique SRF-containing complex formed that was selective for SMCs and, furthermore, that this complex was probably stabilized by protein-protein interactions and not by specific interactions with CArG flanking sequences. Taken together, the results of these studies provide evidence that SM alpha-actin expression in SMCs is complex and may involve the formation of a unique multiprotein initiation complex that is coordinated by SRF complexes bound to multiple CArG elements.

Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions.

The aims of the present studies were to define sufficient promoter sequences required to drive endogenous expression of smooth muscle (SM) alpha-actin and to determine whether regulation of SM alpha-actin expression in vivo is dependent on CArG (CC(A/T)6GG) cis elements. Promoter deletions and site directed mutagenesis techniques were used to study gene regulation in transgenic mice as well as in smooth muscle cell (SMC) cultures. Results demonstrated that a Lac Z transgene that contained 547 bp of the 5' rat SM alpha-actin promoter was sufficient to drive embryonic expression of SM alpha-actin in the heart and in skeletal muscle but not in SMCs. Transient transfections into SMC cultures demonstrated that the conserved CArG element in the first intron had significant positive activity, and gel shift analyses demonstrated that the intronic CArG bound serum response factor. A transgene construct from -2600 through the first intron (p2600Int/Lac Z) was expressed in embryos and adults in a pattern that closely mimicked endogenous SM alpha-actin expression. Expression in adult mice was completely restricted to SMCs and was detected in esophagus, stomach, intestine, lung, and nearly all blood vessels, including coronary, mesenteric, and renal vascular beds. Mutation of CArG B completely inhibited expression in all cell types, whereas mutation of the intronic CArG selectively abolished expression in SMCs, which suggests that it may act as an SMC-specific enhancer-like element. Taken together, these results provide the first in vivo evidence for the importance of multiple CArG cis elements in the regulation of SM alpha-actin expression.

Characterization of graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase.

Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.

Substitution of the degenerate smooth muscle (SM) alpha-actin CC(A/T-rich)6GG elements with c-fos serum response elements results in increased basal expression but relaxed SM cell specificity and reduced angiotensin II inducibility.

We have previously demonstrated that both CC(A/T-rich)6GG (CArG) elements A and B of the smooth muscle (SM) alpha-actin promoter are required for smooth muscle cell (SMC)-specific expression and angiotensin II (AII)-induced stimulation. Moreover, results provided evidence that AII responsiveness of SM alpha-actin was at least partially dependent on modulation of serum response factor (SRF) binding to the SM alpha-actin CArGs by the homeodomain containing protein, MHox. The goal of the present study was to investigate whether the degeneracy of the SM alpha-actin CArGs (both contain a Gua or Cyt substitution in their A/T-rich center) and their reduced SRF binding activity as compared with c-fos serum response element (SRE) is important for conferring cell type-specific expression and AII responsiveness. Transient transfection assays using SM alpha-actin reporter gene constructs in which the endogenous SM alpha-actin CArGs were replaced by c-fos SREs demonstrated the following: 1) relaxation of cell-specific expression, 2) a 50% reduction in AII responsiveness, and 3) reduced ability to be transactivated by MHox. In addition, we also showed that the position of the SM alpha-actin CArGs was important in that interchanging them abolished both basal and AII-induced activities. Taken together, these results suggest that the reduced SRF binding activities of the SM alpha-actin CArGs and CArG positional context contribute to SMC-specific expression of SM alpha-actin as well as maximal AII responsiveness.

Myocardial flavin reductase and riboflavin: a potential role in decreasing reoxygenation injury.

Ferrylmyoglobin has been implicated in cardiac reoxygenation damage. Flavin reductase, an enzyme previously isolated from erythrocytes, can reduce ferrylmyoglobin in the presence of sufficient flavin concentrations. Flavin reductase mRNA signals were detected in rabbit heart, lung, liver, kidney, and isolated cardiomyocytes. It was hypothesized that increasing flavin reductase catalysis by administering flavins exogenously could decrease cardiac reoxygenation damage in isolated rabbit hearts. Riboflavin (150 microM) inhibited reoxygenation-induced lactate dehydrogenase release by 57%, an effect prevented by hematoporphyrin, a flavin reductase inhibitor. The results suggest that riboflavin supplementation has cardioprotective effects during reoxygenation and that these effects are mediated by flavin reductase.

Pyrroloquinoline quinone acts with flavin reductase to reduce ferryl myoglobin in vitro and protects isolated heart from re-oxygenation injury.

Pyrroloquinoline quinone has been isolated from bacteria and recently has been detected in mammalian tissues and fluids. We report in vitro studies which show that pyrroloquinoline quinone serves as a high-affinity substrate for an erythrocyte "flavin reductase" and that the pyrroloquinoline quinol generated by this catalysis reacts rapidly with ferryl myoglobin radical. Western blot analysis of rat and rabbit heart homogenates detects a cross-reactive protein which has a molecular weight identical to the erythrocyte reductase from the same species. Low concentrations of pyrroloquinoline quinone protect isolated rabbit heart from re-oxygenation injury, serving as an effective tissue-protective agent in this model for cellular oxidative damage. We propose that this tissue protection is due to a pyrroloquinoline quinol-mediated reduction of reactive oxygen species.

Ultrastructural demonstration of peroxidative activity and peroxidation in ischaemic and ischaemic-reperfused rabbit hearts.

OBJECTIVE: The aim was to characterise subcellular histochemical evidence of the involvement of peroxidation and peroxidases in myocardial reperfusion injury. The histochemical technique involved the use of 3,3'-diaminobenzidine (DAB), which reacts with peroxides and proteins with peroxidase activity to form an electron dense polymer. METHODS: Isolated rabbit hearts were perfused (Langendorff method) for 30 min with oxygenated physiological saline solution. Some were subjected to 30 min of normothermic global ischaemia, with or without 30 min reperfusion. Non-ischaemic control hearts were perfused continuously for 90 min. Hearts were fixed with glutaraldehyde and cut into 100-150 microns sections that were incubated for 1 h in buffered DAB (1 mg.ml-1) with or without added KCN or H2O2. They were processed further for transmission electron microscopy. Planimetry was done on micrographs taken from random fields (approximately 500 photos). RESULTS: The total amount of DAB polymer in non-ischaemic control heart sections incubated with DAB alone occupied 1.19(SEM 0.44) micron 2 x 1000 micron-2 total cell area. For ischaemic-nonreperfused hearts, the value was 2.32(0.90) micron 2 x 1000 micron-2 (p = 0.223 v control); DAB occupied 7.49(1.42) micron 2 x 1000 micron-2 in ischaemic-reperfused hearts (p = 0.001 v control). DAB positive staining of mitochondria and lipid droplets, but not of peroxisomes, was significantly increased in reperfused hearts compared with non-ischaemic controls. CONCLUSIONS: Reperfusion, but not ischaemia, was associated with increased DAB staining. This suggests a reperfusion induced increase in myocyte peroxidation. Increased staining may be due to the actions of haem proteins with peroxidase activity on peroxidized lipid.

Evidence that NADPH-dependent methemoglobin reductase and administered riboflavin protect tissues from oxidative injury.

NADPH-dependent methemoglobin reductase, first detected in erythrocytes sixty years ago, has subsequently been purified and characterized as a methylene blue reductase and a flavin reductase. The reductase plays no role in methemoglobin reduction under normal conditions, but its activity serves as the basis for the treatment of methemoglobinemia with methylene blue or flavin. On-going studies demonstrate that this cytosolic protein is also present in liver and that its primary structure distinguishes it from other known proteins. The bovine erythrocyte reductase tightly binds hemes, porphyrins, and fatty acids with resulting loss of activity. Pyrroloquinoline quinone serves as a high-affinity substrate of the reductase, suggesting that this naturally-occurring compound may be a physiological substrate. The ability of the reductase to catalyze the intracellular reduction of administered riboflavin to dihydroriboflavin suggested that this system might be exploited to protect tissues from oxidative damage. This hypothesis was supported by our finding that dihydroriboflavin reacts rapidly with Fe(IV)O and Fe(V)O oxidation states of hemeproteins, states that have been implicated in tissue damage associated with ischemia and reperfusion. Preliminary studies demonstrate that, as predicted, administration of low concentrations of riboflavin protects isolated rabbit heart from reoxygenation injury, rat lung from injury resulting from systemic activation of complement, and rat brain from damage caused by four hours of ischemia. Data from these animal studies suggest that flavin therapy holds promise in protecting tissue from the oxidative injuries of myocardial infarction, acute lung injury, stroke, and a number of other clinical conditions.