br Materials and Methods br

Materials and Methods
Results
Discussion 15-HETE is a predominant eicosanoid in pulmonary vascular remodeling, and it could promote hypoxia-induced PAH via its involvement in the regulation of proliferation and cell cycle progression in PASMCs. To understand its role in HPH, we have previously shown that the proliferation and cell cycle progression induced by 15-HETE requires the induction of expression of ERK1/2, MAPK, ROCK, and growth factors [6], [8], [13], [14], [15], [16]. AP-1 has been reported to be involved in the progression of proliferation in multiple cancer cell lines. The finding that AP-1 knockdown prevents the 15-HETE-induced proliferation and cell cycle progression suggests that AP-1 may be involved in 15-HETE-mediated hypoxia-triggered PAH. Additionally, the activation of AP-1 feedback regulates the expression of 15-LOX. Thus, the present study indicates a reciprocal regulation between AP-1 and 15-LOX/15-HETE signaling, which contributes to the hypoxia-induced PASMC phenotype alteration, including proliferation and cell cycle progression. Both 15-LOX1 and 15-LOX2 convert arachidonic A 804598 to 15-HETE as a predominant eicosanoid under hypoxia condition. We have previously shown that 15-HETE stimulates pulmonary artery constriction as well as vascular remodeling under hypoxia condition [4], [5], [6], [13]. It is well accepted that the pulmonary artery vascular remodeling is primarily due to the proliferation of PASMCs, which leads to medial hypertrophy of the vascular wall. We have shown that 15-HETE-induced PASMC proliferation requires activation of ERK1/2 [16], MAPK [8], and ROCK pathway [6]. AP-1 has been reported to be involved in the progression of cell proliferation and to be critical in tumor growth and metastasis as well [10], [12]. In particular, c-Fos and c-Jun have been shown to be expressed above basal levels in response to the introduction of growth factors, such as TGF alpha, TGF beta, and IL2 in the cell [17]. The finding that hypoxia/15LOX/15-HETE axis increases the expression of c-jun and c-fos, which possess the capacity to induce proliferation and cell cycle transition to S+G2/M phase, suggests that 15-HETE (in addition to other growth factors) serves as an important mediator in promoting PASMC proliferation via its activation by AP-1. It has been shown that 15-HETE activates JNK1 in human retinal microvascular endothelial cells [18]. The expression of the Fos and Jun family of AP-1 transcription factors requires the activation of the Rac1-MEK1-JNK1 axis [19]. It is logical to assume that AP-1 constitutes a signal for hypoxia/15-HETE-induced PASMC proliferation. Further study is needed to examine the signaling pathway involved in 15-HETE-activated AP-1 expression. The effects of hypoxia on AP-1 expression have been controversial. Ausserer and coworkers reported that hypoxia (6 h) increased c-jun expression in human carcinoma cells [20], whereas Jin et al. have shown that exposure to hypoxia did not stimulate but downregulated c-jun and c-fos expression in pulmonary arteries [21]. The different observations of hypoxia-induced altered expressions of c-jun and c-fos are not clear. One possible reason could be the different response to hypoxia of the two species of rats (Wistar vs. Sprague Dawley) used. Another reason could be that the detection protocol used was different, e.g., immunoprecipitation was performed whereby the protein may undergo degradation during the process. In this study, we detected that 15-LO/15-HETE is medicated the increasing AP-1 expression induced by hypoxia. We also observed 5-hydroxyeicosatetraenoic acid (5-HETE) increased the expression of AP-1 in PASMCs (Data not shown). In consistently, Maayah et al observed a similar effect of 5-HETE, 12-HETE and 15-HETE in cellular hypertrophy through mitogen-activated protein kinase and nuclear factor kappa B dependent mechanism [22]. However, 20- hydroxyeicosatetraenoic acid inhibited AP-1 expression by activated the AKT/GSK-3β pathway in rat insulinoma INS-1E cells [23]. The controversial effect of monohydroxy arachidonic acid on AP-1 expression still need to elucidate in further study.