Monocrotaline pyrrole induces Smad nuclear accumulation and altered signaling expression in human pulmonary arterial endothelial cells - PubMed (original) (raw)

Monocrotaline pyrrole induces Smad nuclear accumulation and altered signaling expression in human pulmonary arterial endothelial cells

M Ramos et al. Vascul Pharmacol. 2007 Jun.

Abstract

The mechanistic relationship between the widely used monocrotaline model of primary pulmonary hypertension and altered TGFbeta family signaling due to genetic defects in the Bone Morphogenetic Protein type II receptor in affected humans has not been investigated. In this study we use fluorescent microscopy to demonstrate nuclear translocation of Smad 4 in human pulmonary arterial endothelial cell (HPAEC) cultures treated with monocrotaline pyrrole (MCTP), Bone Morphogenetic Protein (BMP) and TGFbeta. While MCTP induced transient nuclear accumulation of phosphorylated Smad 1 (P-Smad 1) and phosphorylated Smad 2 (P-Smad 2), only expression of P-Smad 1 was significantly altered in western blots. P-Smad 1 expression significantly increased 30 min following treatment with MCTP correlating with P-Smad 1 and Smad 4 nuclear translocation. Although a modest, but significant decrease in P-Smad 1 expression occurred 1 h after treatment, expression was significantly increased at 72 h. Evaluation of components of the signal and response pathway at 72 h showed decreased expression of the BMP type II receptor (BMPrII), no change in TGFbeta Activin Receptor-like Kinase 1 (Alk 1), no change in Smad 4 but increase in the inhibitory Smad 6, decrease in the alternate BMP signaling pathway p38(MAPK) but no change in the psmad1 response element ID 1. Our results suggest transient activation of Smad signaling pathways in initial MCTP endothelial cell toxicity, and a persistent dysregulation of BMP signaling. Electron microscopy of cell membrane caveoli revealed a dramatic decrease in these structures after 72 h. Loss of these structural elements, noted for their sequestration and inhibition of receptor activity, may contribute to prolonged alterations in BMP signaling.

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Figures

Figure 1

Smad 4 nuclear translocation in HPAEC treated with 60 μg/ml MCTP: A) Non-treated control cells. Smad 4 is represented by fluorescent label that is scattered predominantly throughout the cytoplasm. The nuclei appear as open vacuoles, containing little signal. B) Cells treated 15 min. There is an increase in fluorescence around and within the nuclei, while the peripheral cytoplasm contains scattered fluorescent signal. C) Cells treated 30 min. Smad 4 is concentrated within the nuclei, which appear as intensely fluorescent green balls. D) Cells treated for 60 min. The signal is predominantly cytoplasmic in the majority of the cells. Bars = 50 μm.

Figure 2

Smad 4 nuclear translocation in HPAEC treated for 1 hour with recombinant ligands: A) Cells treated with 100 ng/ml BMP4. B) Cells treated with 300 ng/ml BMP2. C) Cells treated with 20 ng/ml TGFβ-1. D) Results of nuclear counts show intense nuclear signal was less frequently observed in ligand-treated cells than cells treated with MCTP. Bars = 50 μm.

Figure 3

Translocation of endogenous Smad 4 in HEK293H cells in cells treated with MCTP or BMP4 1 hr: A) Non-treated cells. B) Cells treated with 60 μg/ml MCTP. C) Cells treated with 100 ng/ml BMP4. Nuclear translocation was greater in treated cells. Bars = 50 μm.

Figure 4

Translocation of transfected GFP-Smad 4 in HEK293H cells treated with 60μg/ml MCTP: A) Non-treated cells, and B) Cells treated for 1 hr. Bars = 50 μm. Images were converted to grey-scale, and qualitative analysis of nuclear translocation C) was based on pixel intensity, which was greater in MCTP-treated cells (* p<.05).

Figure 5

Nuclear translocation of P-Smad 1 in HPAEC treated with MCTP: A) Non-treated control cells. Signal is scattered throughout the cytoplasm, and is present within the nuclei of most cells. B) Cells treated 30 min with MCTP. In contrast to the cells pictured in “A”, the intensity of the signal is concentrated within the nuclei of most cells present. C) Cells after 60 min. Nuclear signal has diminished. (Similar results were obtained with P-Smad 2.) Bars = 50 μm.

Figure 6

Western Blot of P-Smad 1 and P-Smad 2 in DMF vehicle (C1–C4) vs. 60 μg/ml MCTP (M1–M4) treated cells, 30 min: A) P-Smad 1 is significantly increased in cells treated for 30 min. with MCTP (_p_= 0.008) By 1 hr, P-Smad 1 is significantly decreased in MCTP treated cells but by 72 hr. P-Smad 1 is again significantly increased (_p_=0.018). B) There was no significant difference in expression of P-Smad 2 at any time point. All lanes contain 50 μg protein. C) Densitometric analysis of western blots (* p<.05).

Figure 7

Western Blot of HPAEC 72 hr treatment; DMF vehicle (C1–C3) vs. 60 μg/ml MCTP (M1–M3): A) Expression of BMPrII is significantly decreased in MCTP treated cells (_p_= 0.05). No significant change was present in treated cells for B) Alk1 (_p_= 0.07) or C) Smad 4. D) Smad 6 expression is significantly increased (_p_= 0.035) while E) Phospho-p38 MAPK is significantly decreased (_p_= 0.012). No change was evident in F) ID 1 (_p_= 0.51) or G) Cav-1 (_p_= 0.58). All lanes contain 50 μg protein. G) Densitometric analysis of 72 hr. western blots (* p<.05).

Figure 8

EM of HPAEC depicting caveolae, indicated by an asterisk: A) Non-treated control cell. Caveolae are present on both apical and basal surfaces of the peripheral edge of this cell. B) Cell treated 48 hr with MCTP. Caveolae are located at both the membrane and in clusters just below the surface. C, D, & E) Cells treated for 72 hr with MCTP. Caveolae are sparsely distributed on the cell membrane. F) Cell treated 72 hr with DMF vehicle. Numerous caveolae are present both on the cell membrane and within the cytoplasm. Bars = 500 nm.

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Monocrotaline pyrrole induces Smad nuclear accumulation and altered signaling expression in human pulmonary arterial endothelial cells - PubMed (original) (raw)