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Transforming growth factor-b (TGF-b) is a cytokine implicated in wound healing and in the pathogenesis of pulmonary fibrosis. TGF-b stimulates myofibroblast differentiation characterized by expression of contractile smooth muscle (SM)-specific proteins such as SM-a-actin. In the present study, we examined the role of serum response factor (SRF) in the mechanism of TGF-b-induced pulmonary myofibroblast differentiation of human lung fibroblasts (HLF). TGF-b stimulated SM-a-actin expression in HLF, which paralleled with a profound induction of SRF expression and activity. Inhibition of SRF by the pharmacologic SRF inhibitor (CCG-1423), or via adenovirus-mediated transduction of SRF short hairpin RNA (shSRF), blocked the expression of both SRF and SM-a-actin in response to TGF-b without affecting Smad-mediated signaling of TGF-b. However, forced expression of SRF on its own did not promote SM-a-actin expression, whereas expression of the constitutively transactivated SRF fusion protein (SRF-VP16) was sufficient to induce SM-a-actin expression, suggesting that both expression and transactivation of SRF are important. Activation of protein kinase A (PKA) by forskolin or iloprost resulted in a significant inhibition of SMa-actin expression induced by TGF-b, and this was associated with inhibition of both SRF expression and activity, but not of Smadmediated gene transcription. In summary, this is the first direct demonstration that TGF-b-induced pulmonary myofibroblast differentiation is mediated by SRF, and that inhibition of myofibroblast differentiation by PKA occurs through down-regulation of SRF expression levels and SRF activity, independent of Smad signaling.

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Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate beta-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). beta-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of beta-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous beta-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) beta-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous beta-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.

Abstract-Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na ϩ ]/[K ϩ ] ratio after the sustained blockage of the Na ϩ ,K ϩ -ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na Key Words: apoptosis Ⅲ vascular smooth muscle Ⅲ proteome Ⅲ ion transport Ⅲ ouabain R emodeling of the blood vessel plays an important role in a variety of human vascular disorders, including hypertension, 1-3 atherosclerosis, 4 arterial injury, and restenosis after angioplasty. 5-7 Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as the main factor contributing to the regulation of their number during remodeling, 8 -12 which inspired numerous studies of the mechanisms of the induction and progression of VSMC apoptosis. The execution phase of apoptosis in VSMCs is triggered similarly to that in the other cell types by activation of the caspase cascade, cleavage of intracellular proteins, and final disintegration of the cell. In contrast, the induction phase is specific for different subtypes of remodeling and involves the integration of multiple pro-and antiapoptotic signals, including the expression of death receptors, protooncogenes, and tumor suppressor genes. [13][14][15][16][17][18][19] Our recent studies showed that inhibition of the VSMC Na ϩ ,K ϩ pump with ouabain, or in K ϩ -free medium, rescues cells from apoptosis triggered by a number of factors including serum deprivation. 20 Equimolar substitution of extracellular Na ϩ with K ϩ completely abolished the effect of ouabain 20 showing that antiapoptotic action was indeed caused by the inversion of [NaThe development of cell death was blocked upstream of caspase-3 activati...

The growing research interest in nanomedicine for the treatment of cancer and inflammatory-related pathologies is yielding encouraging results. Unfortunately, enthusiasm is tempered by the limited specificity of the enhanced permeability and retention effect. Factors such as lack of cellular specificity, low vascular density, and early release of active agents prior to reaching their target contribute to the limitations of the enhanced permeability and retention effect. However, improved nanomedicine designs are creating opportunities to overcome these problems. In this review, we present examples of the advances made in this field and endeavor to highlight the potential of these emerging technologies to improve targeting of nanomedicine to specific pathological cells and tissues.