HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1α) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX−/−/BAK−/− murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.

BACKGROUND AND PURPOSE Acute silencing of caveolin‐1 (Cav‐1) modulates receptor‐mediated contraction of airway smooth muscle. Moreover, COX‐2‐ and 5‐lipoxygenase (5‐LO)‐derived prostaglandin and leukotriene biosynthesis can influence smooth muscle reactivity. COX‐2 half‐life can be prolonged through association with Cav‐1. We suggested that lack of Cav‐1 modulated levels of COX‐2 which in turn modulated tracheal contraction, when arachidonic acid signalling was disturbed by inhibition of COX‐2.EXPERIMENTAL APPROACH Using tracheal rings from Cav‐1 knockout (KO) and wild‐type mice (B6129SF2/J), we measured isometric contractions to methacholine and used PCR, immunoblotting and immunohistology to monitor expression of relevant proteins.KEY RESULTS Tracheal rings from Cav‐1 KO and wild‐type mice exhibited similar responses, but the COX‐2 inhibitor, indomethacin, increased responses of tracheal rings from Cav‐1 KO mice to methacholine. The phospholipase A2 inhibitor, eicosatetraynoic acid, which inhibits formation of both COX‐2 and 5‐LO metabolites, had no effect on wild‐type or Cav‐1 KO tissues. Indomethacin‐mediated hyperreactivity was ablated by the LTD4 receptor antagonist (montelukast) and 5‐LO inhibitor (zileuton). The potentiating effect of indomethacin on Cav‐1 KO responses to methacholine was blocked by epithelial denudation. Immunoprecipitation showed that COX‐2 binds Cav‐1 in wild‐type lungs. Immunoblotting and qPCR revealed elevated levels of COX‐2 and 5‐LO protein, but not COX‐1, in Cav‐1 KO tracheas, a feature that was prevented by removal of the epithelium.CONCLUSION AND IMPLICATIONS The indomethacin‐induced hypercontractility observed in Cav‐1 KO tracheas was linked to increased expression of COX‐2 and 5‐LO, which probably enhanced arachidonic acid shunting and generation of pro‐contractile leukotrienes when COX‐2 was inhibited.

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β1. As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β1 induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β1. The failure by TGF-β1 to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β1 signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.

BACKGROUND:Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.METHODS:We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).RESULTS:Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.CONCLUSIONS:We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

Geranylgeranyl transferase 1 (GGT1) is involved in the posttranslational prenylation of signaling proteins, such as small GTPases. We have shown that blocking the formation of isoprenoids with statins regulates survival of human lung mesenchymal cells; thus, we tested the hypothesis that GGT1 may specifically modulate programmed cell death pathways in these cells. To this end, human airway smooth muscle (HASM) cells were treated with the selective GGT1 inhibitor GGTi-298. Apoptosis was seen using assays for cellular DNA content and caspase activation. Induction of autophagy was observed using transmission electron microscopy, immunoblotting for LC3 lipidation and Atg5-12 complex content, and confocal microscopy to detect formation of lysosome-localized LC3 punctae. Notably, GGT1 inhibition induced expression of p53-dependent proteins, p53 upregulated modulator of apoptosis (Noxa), and damage-regulated autophagy modulator (DRAM), this was inhibited by the p53 transcriptional activation inhibitor cyclic-pifithrin-α. Inhibition of autophagy with bafilomycin-A1 or short-hairpin RNA silencing of Atg7 substantially augmented GGTi-298-induced apoptosis. Overall, we demonstrate for the first time that pharmacological inhibition of GGT1 induces simultaneous p53-dependent apoptosis and autophagy in HASM. Moreover, autophagy regulates apoptosis induction. Thus, our findings identify GGT1 as a key regulator of HASM cell viability.

The dystrophin–glycoprotein complex (DGC) links the extracellular matrix and actin cytoskeleton. Caveolae form membrane arrays on smooth muscle cells; we investigated the mechanism for this organization. Caveolin-1 and β-dystroglycan, the core transmembrane DGC subunit, colocalize in airway smooth muscle. Immunoprecipitation revealed the association of caveolin-1 with β-dystroglycan. Disruption of actin filaments disordered caveolae arrays, reduced association of β-dystroglycan and caveolin-1 to lipid rafts, and suppressed the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release. We generated novel human airway smooth muscle cell lines expressing shRNA to stably silence β-dystroglycan expression. In these myocytes, caveolae arrays were disorganized, caveolae structural proteins caveolin-1 and PTRF/cavin were displaced, the signaling proteins PLCβ1 and Gαq, which are required for receptor-mediated Ca2+ release, were absent from caveolae, and the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release, was diminished. These data reveal an interaction between caveolin-1 and β-dystroglycan and demonstrate that this association, in concert with anchorage to the actin cytoskeleton, underpins the spatial organization and functional role of caveolae in receptor-mediated Ca2+ release, which is an essential initiator step in smooth muscle contraction.

Statins inhibit 3-hydroxy-3-methyl-glutarylcoenzyme CoA (HMG-CoA) reductase, the proximal enzyme for cholesterol biosynthesis. They exhibit pleiotropic effects and are linked to health benefits for diseases including cancer and lung disease. Understanding their mechanism of action could point to new therapies, thus we investigated the response of primary cultured human airway mesenchymal cells, which play an effector role in asthma and chronic obstructive lung disease (COPD), to simvastatin exposure. Simvastatin induced apoptosis involving caspase-9, -3 and -7, but not caspase-8 in airway smooth muscle cells and fibroblasts. HMG-CoA inhibition did not alter cellular cholesterol content but did abrogate de novo cholesterol synthesis. Pro-apoptotic effects were prevented by exogenous mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, downstream products of HMG-CoA. Simvastatin increased expression of Bax, oligomerization of Bax and Bak, and expression of BH3-only p53-dependent genes, PUMA and NOXA. Inhibition of p53 and silencing of p53 unregulated modulator of apoptosis (PUMA) expression partly counteracted simvastatin-induced cell death, suggesting a role for p53-independent mechanisms. Simvastatin did not induce mitochondrial release of cytochrome c, but did promote release of inhibitor of apoptosis (IAP) proteins, Smac and Omi. Simvastatin also inhibited mitochondrial fission with the loss of mitochondrial Drp1, an essential component of mitochondrial fission machinery. Thus, simvastatin activates novel apoptosis pathways in lung mesenchymal cells involving p53, IAP inhibitor release, and disruption of mitochondrial fission.

Tumor necrosis factor alpha (TNF) is a cytokine that induces caspase-dependent (apoptotic) and caspase-independent (necrosis-like) cell death in different cells. We used the murine fibrosarcoma cell line model L929 and a stable L929 transfectant over-expressing a mutated dominant-negative form of BNIP3 lacking the C-terminal transmembrane (TM) domain (L929-ΔTM-BNIP3) to test if TNF-induced cell death involved pro-apoptotic Bcl2 protein BNIP3. Treatment of cells with TNF in the absence of actinomycin D caused a rapid fall in the mitochondrial membrane potential (ΔΨm) and a prompt increase in reactive oxygen species (ROS) production, which was significantly less pronounced in L929-ΔTM-BNIP3. TNF did not cause the mitochondrial release of apoptosis inducing factor (AIF) and Endonuclease G (Endo-G) but provoked the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 at similar levels in both L929 and in L929-ΔTM-BNIP3 cells. We observed TNF-associated increase in the expression of BNIP3 in L929 that was mediated by nitric oxide and significantly inhibited by nitric oxide synthase inhibitor N5-(methylamidino)-l-ornithine acetate. In L929, lysosomal swelling and activation were markedly increased as compared to L929-ΔTM-BNIP3 and could be inhibited by treatment with inhibitors to vacuolar H+-ATPase and cathepsins −B/−L. Together, these data indicate that TNF-induced cell death involves BNIP3, ROS production, and activation of the lysosomal death pathway.