Transforming growth factor-β1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells

Abstract

Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Pulmonary smooth muscle cells and fibroblasts: NOX4-Studies in pulmonary fibroblasts and smooth muscle cells revealed the expression of p47phox, p67phox, p22phox and NOX4, and demonstrated increased production of ROS in response to inflammatory mediators, such as tumor necrosis factor β (TNF-β) or transforming growth factor β (TGF-β), which is primary associated with selective upregulation of NOX4 (185)(186)(187)(188). In addition, NOX4 expression is also induced by other stimuli, including hypoxia/ischemia, shear stress, and ER stress, but to date no information appears to be available regarding the NOX4 promoter or its transcriptional regulators (6,16,81).…”

“…Studies in isolated smooth muscle cells indicated that NOX4 expression in freshly isolated cells rapidly declines with multiple passages, in close association with loss of smooth muscle differentiation markers such as smooth muscle α-actin (SMA), myosin heavy chain (186,190). Subsequent studies with NOX4-targeted siRNA demonstrated that NOX4 is required for the expression of smooth muscle differentiation markers, and maintenance of SMA-based stress fibers (190).…”

“…For example, NOX4-mediated ROS production in human pulmonary artery smooth muscle cells in response to TGF-β occurs primarily intracellularly, and NOX4 appears to localize to the ER or the nucleus (186). Accordingly, NOX4 activation was associated with intracellular ERK1/2 signaling and phosphorylation of nuclear or ER proteins, such as retinoblastoma protein and eukaryotic translation initiation factor 4E binding protein-1, thereby mediating smooth muscle cell proliferation and hypertrophy (186,193).…”

“…For example, NOX4-mediated ROS production in human pulmonary artery smooth muscle cells in response to TGF-β occurs primarily intracellularly, and NOX4 appears to localize to the ER or the nucleus (186). Accordingly, NOX4 activation was associated with intracellular ERK1/2 signaling and phosphorylation of nuclear or ER proteins, such as retinoblastoma protein and eukaryotic translation initiation factor 4E binding protein-1, thereby mediating smooth muscle cell proliferation and hypertrophy (186,193). In contrast, myofibroblasts obtained from patients with iodopathic pulmonary fibrosis were found to produce primarily extracellular H 2 O 2 in response to TGF-β1, and are thereby capable of inducing cell death in neighboring pulmonary epithelial cells, suggesting the association of NOX4 with the plasma membrane as a paracrine signaling molecule (187).…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…The Nox4 isoform has been generally considered to be constitutively active. It was recently found that the insulin receptor [156], toll-like receptor 4 [125], transforming growth factor β1 [145,146], and possibly interleukin 1β [39,57,59] all required Nox4 activity. In adipocytes, overexpression of Nox4 was not sufficient to increase basal levels of ROS but required insulin stimulation, arguing for a regulated Nox4 activity.…”