title: Transforming growth factor-beta1 causes pulmonary microvascular endothelial cell apoptosis via ALK5.
abstract: We have previously shown that transforming growth factor (TGF)-beta1 protected against main pulmonary artery endothelial cell (PAEC) apoptosis induced by serum deprivation and VEGF receptor blockade through a mechanism associated with ALK5-mediated Bcl-2 upregulation. In the current study, we investigated the effect of TGF-beta1 on pulmonary microvascular endothelial cell (PMVEC) apoptosis. We found that, in contrast to the results seen in conduit PAEC, TGF-beta1 caused apoptosis of PMVEC, an effect that was also dependent on ALK5 activity. We noted that non-SMAD signaling pathways did not play a role in TGF-beta1-induced apoptosis. Both SMAD2 and SMAD1/5 were activated upon exposure to TGF-beta1. TGF-beta1-induced activation of SMAD2, but not SMAD1/5, was abolished by ALK5 inhibition, an effect that associated with prevention of TGF-beta1-induced apoptosis. These results suggest that SMAD2 is important in TGF-beta1-induced apoptosis of PMVEC. While caspase-12 activity was not altered, caspase-8 was activated by TGF-beta1, an effect that correlated with a reduction of cFLIP protein levels. Additionally, TGF-beta1 decreased Bcl-2 protein levels and induced cytochrome c cytosolic redistribution. These results suggest that TGF-beta1 caused apoptosis of PMVEC likely through both caspase-8-dependent extrinsic pathway and mitochondria-mediated intrinsic pathway. We noted that inhibition of ALK5 attenuated serum deprivation-induced apoptosis, an effect that correlated with increased expression and activation of CREB and its potential target genes, Bcl-2 and cFLIP. These results suggest that CREB may be important in mediating apoptosis resistance of PMVEC upon ALK5 inhibition perhaps through upregulation of Bcl-2 and cFLIP. Finally, we noted that SMAD1/5 were activated upon ALK5 inhibition in the presence of low levels of TGF-beta1, an effect associated with enhanced endothelial proliferation. We speculate that imbalance of ALK1 and ALK5 may contribute to the development of pulmonary artery hypertension.
authors.affiliation: Providence VA Medical Center, Research Services, Providence, RI 02908, USA. Qing_Lu@brown.edu
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
authors.forename: Bhuvic
authors.initials: B
authors.lastname: Patel
authors.forename: Elizabeth O
authors.initials: EO
authors.lastname: Harrington
authors.forename: Sharon
authors.initials: S
authors.lastname: Rounds
mesh_terms: D029404:Activin Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000098:blood supply* / Q000166:cytology*; D055806:Microvessels / Q000166:cytology*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D017362:Cyclic AMP Response Element-Binding Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; D029404:Activin Receptors; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human; C000625843:Tgfbr1 protein, rat; D053181:Caspase 8