PLoS ONE. 2015 Dec 28;10(12):e0145523.

TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells

Eiselein L1, Nyunt T1, Lamé MW2, Ng KF1, Wilson DW3, Rutledge JC1, Aung HH1.
  • 1Department of Internal Medicine, Division of Cardiovascular Medicine, School of Medicine, University of California Davis, Davis, California, 95616, United States of America.
  • 2Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis, California, 95616, United States of America.
  • 3Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California Davis, Davis, California, 95616, United States of America.



Studies have suggested a link between the transforming growth factor beta 1 (TGF-β1) signaling cascade and the stress-inducible activating transcription factor 3 (ATF3). We have demonstrated that triglyceride-rich lipoproteins (TGRL) lipolysis products activate MAP kinase stress associated JNK/c-Jun pathways resulting in up-regulation of ATF3, pro-inflammatory genes and induction of apoptosis in human aortic endothelial cells. Here we demonstrate increased release of active TGF-β at 15 min, phosphorylation of Smad2 and translocation of co-Smad4 from cytosol to nucleus after a 1.5 h treatment with lipolysis products. Activation and translocation of Smad2 and 4 was blocked by addition of SB431542 (10 μM), a specific inhibitor of TGF-β-activin receptor ALKs 4, 5, 7. Both ALK receptor inhibition and anti TGF-β1 antibody prevented lipolysis product induced up-regulation of ATF3 mRNA and protein. ALK inhibition prevented lipolysis product-induced nuclear accumulation of ATF3. ALKs 4, 5, 7 inhibition also prevented phosphorylation of c-Jun and TGRL lipolysis product-induced p53 and caspase-3 protein expression. These findings demonstrate that TGRL lipolysis products cause release of active TGF-β and lipolysis product-induced apoptosis is dependent on TGF-β signaling. Furthermore, signaling through the stress associated JNK/c-Jun pathway is dependent on TGF-β signaling suggesting that TGF-β signaling is necessary for nuclear accumulation of the ATF3/cJun transcription complex and induction of pro-inflammatory responses.

PMID: 26709509



Elevation of blood triglyceride-rich lipoproteins (TGRL) is a known atherosclerotic cardiovascular disease risk factor and can induce endothelial dysfunction and inflammation. TGRL include chylomicrons, which contain triglycerides derived from the exogenous (intestine-derived) pathway, and VLDL (very low-density lipoprotein), which contain triglycerides from the endogenous (liver-derived) pathway. Lipolysis of TGRL occurs when lipoproteins bind to lipoprotein lipase (LpL) (1), an enzyme anchored to the surface of endothelial cells (2, 3). Increased concentrations of TGRL lipolysis products (lipolysis) stimulate the expression of multiple proinflammatory, procoagulant, and proapoptotic genes in cultured endothelial cells (4-6). Lipolysis releases neutral and oxidized free fatty acids (FFAs) that activate endothelial cell (EC) inflammation in high physiological and pathophysiological concentrations and injure endothelial cells by increasing VLDL remnant deposition in the artery wall (7, 8). Lipolysis products augment endothelial monolayer permeability, perturb cell junction and contractile proteins zonula occludens-1 and F-actin, and induce apoptosis (9). Lipolysis products also significantly increase the production of reactive oxygen species (ROS) in EC and alter lipid raft morphology (7). We recently reported that lipolysis of human post-prandial blood lipids activate MAPK/Janus Kinase (JNK) stress response pathways that induce expression of multiple pro-inflammatory and pro-apoptotic genes leading to endothelial dysfunction (10). We identified transcription of Activating transcription factor 3 (ATF3), a member of the CREB family of transcription factors, as a key response gene after treatment with lipolysis products and demonstrated that its induction was essential for the expression of a subset of pro-inflammatory responses in human aortic endothelial cells.


ATF3 is expressed ubiquitously at a low level in the absence of cellular stresses(11). Upon exposure to various conditions, such as hypoxia, DNA damaging agents (etoposide, ionizing radiation, UV irradiation), heat shock, cold shock, nutrient starvation, and serum stimulation, ATF3 is rapidly induced by transcriptional activation. ATF3 proteins are localized in the nucleus. ATF3 is one of immediate early response genes and plays role in determining cell fate. Studies, mostly carried out in vitro using established cancer cell lines, show that ATF3 has dual functions in promoting either cell death or cell survival under different conditions. These cell and context specific responses likely relate to differential binding to transcription factor complexes including the AP-1 complex transducing the stress associated JNK pathway.


Studies have shown that in vascular EC, TGF-β1 is a potent inducer of apoptosis. TGF-β1 is released from a secreted latent complex and is involved in many different biological processes, such as cell growth, cell cycle progression, migration, differentiation, matrix production and apoptosis (12). Recent studies have suggested that ATF3 up-regulation and activity as a transcription factor can be controlled by, and depends on, prior TGFβ receptor activation. TGF-β stimulation has been shown to rapidly induce the expression of ATF3 via a Smad3 containing transcriptional complex (13). Furthermore, ATF3 has been shown to directly interact and complex with Smad3 and Smad4 to act as a transcriptional repressor. Our prior studies demonstrating lipolysis products induce EC apoptosis suggested a potential role for TGF-β in the response.


We hypothesized that the observed lipolysis-induced up-regulation and activation of ATF3 in vascular endothelial cells is regulated by the TGF-β signaling system and asked whether there is an interface between TGF-β signaling and JNK activation.


In our study, we used human TGRL isolated from healthy human volunteers. Postprandial blood samples were obtained 3.5 h after consumption of a moderately high fat meal, which corresponds to the peak elevation in plasma triglyceride concentrations.

We evaluated signaling pathways by treating cultured human aortic endothelial cells (HAEC) in vitro with lipolysed postprandial TGRL and compared responses with media alone lipoprotein lipase alone or untreated lipid. The rate of TGF-β1 release was significantly increased for cells treated with lipolysis products compared to all other treatments at 15 min (Fig 1).




Figure 1. TGRL lipolysis products release TGF-β1 at 15 min. The rate of TGF-β1 release is significantly increased for cells treated with TGRL (150 mg/dL) + LpL (2 U/mL) (TL) compared to cells treated with Media (M) or LpL alone (L) or TGRL alone (T), at 15 min. Addition of 10 μM ALK to TL (TL+ALK) suppressed TGF-β1 released by TL. N=4/treatment group, P≤0.05 as significant, *= TL compared to M, L, T or TL, #= TL+ALK compared to TL. TGF-β1 was not detected in M, T or TL only, in the absence of cells.


To evaluate lipolysis activate TGF-β family second messenger signaling, we determined Smad2 phosphorylation and Smad4 expression. Our study demonstrates that lipolysis product-mediated release of TGF-β1 specifically activates TGF-β receptors leading to Smad2 phosphorylation. We also show that an inhibitor of the TGF-β type I receptor almost completely abrogates the induced phosphorylation of Smad2 and, blocks the translocation of Smad4 to the nucleus (Fig 2).




Figure 2. Smad2 phosphorylation at 1.5 h and Smad4 nucleus accumulation at 3 h. A) Smad2 and Phospho-Smad2 protein. Western Blot (a) and densitometry quantification (b) lysates from HAEC treated with TL for 1.5 h (TL1.5) show significantly increased Smad2 phosphorylation compared to cells treated with M, T, L or TL for 0.5 h (TL0.5). Addition of 10 μM ALK, TGF-β receptor inhibitor, effectively blocks Smad2 phosphorylation (TL1.5+ALK). N=3/treatment group, P≤0.05 as significant, *=TL1.5 compared to M, L, T, or TL0.5, #=TL1.5+ALK compared to TL1.5. B) (a) HAEC monolayers show changes in localization of Smad4 from cytosol to nucleus after treatment with TL compared to controls treated with M, L or T at 1 h. Treatment with lipolysis products with 10 μM ALK shows partial abrogation of Smad4 translocation. (b) % Accumulation of Smad4 based on counts of fluorescent nuclei. Accumulation was significantly increased after 1 h of treatment with lipolysis (TL1h). The addition 10 μM of inhibitor ALK (TL3+ALK) significantly reduced the observed accumulation. N=5 coverslips/treatment group, P≤0.05, *=TL1, TL2, TL3 compared to M, #=TL3+ALK compared to TL3 (Bar = 20 μm).


We also evaluated the expression of p53, a tumor suppressor protein and regulator of apoptosis, and apoptotic cascade Caspase-3 activity.  Our findings demonstrate that for lipolysis product-induced apoptosis in HAEC cultures, activation of TGF-β receptors is required for p53 up-regulation and apoptosis as measured by caspase-3 activation.


To determine the relevance of our in vitro findings to intact arteries, we performed a similar experiment by perfusing mouse arteries in situ. Our findings demonstrate the induction of apoptosis by lipolysis products in intact arteries. In addition, perfusion of TGRL alone induced a significant percentage (65%) of apoptotic cells suggesting that LpL expressed in endothelium from intact arteries is sufficient to elicit lipolysis product induced signaling. TGRL lipolysis product activates apoptosis both in vitro and in vivo (Fig 3).




Figure 3. TGRL lipolysis products activates apoptosis both in vitro and vivo. A) Lipolysis (TL) increases expression of the active fragment of caspase-3 activity compared to M, L or T in HAEC at 3h. B) Caspase-3 protein expression was blocked by ALK. Additional treatment with 10 μM ALK (TL+ALK) abrogated the expression of the active Caspase-3 fragment. N=3/treatment group, P≤0.05,*=TL compared to TL+ALK. C) Lipolysis activates apoptosis in mouse carotid artery. TUNEL staining of mouse carotid artery. (b) Percentage of apoptosis of endothelial cells based on FITC and nuclear staining. TL significantly induced apoptosis (65%) compare to control in mouse carotid artery. Positive control: DNAse I treated. N=4 mice/group, P≤0.05 as significant, *=T or TL compare to media control group, #=TL compare to T. Magnification=60X, Bar=20 μm.


The present study was designed to determine whether TGRL lipolysis products induce ATF3 via the TGF-β1 signaling cascade, we performed mRNA and protein expression assays. Our data demonstrate that the lipolysis product-induced up-regulation of ATF3, both at the mRNA and the protein level, is dependent on prior activation of the TGF-β receptor. While treatment with lipolysis products alone elicited a strong up-regulation of ATF3, pre-treatment with an inhibitor of the activity of TGF-β1 activin receptor-like kinases (ALKs 4, 5, and 7). Similarly, anti-TGF-β antibody also inhibited this response demonstrating a dependence on both TGF-β and its receptor.


In summary, we have made novel observations that TGRL lipolysis products stimulate release of active TGF-β1 and autocrine activation of the Smad signaling cascade in endothelial cells. We confirm that lipolysis of postprandial lipids induces the up-regulation of the stress protein ATF3 in human aortic endothelial cells and this up-regulation is under the regulatory control of the TGF-β/Smad signaling cascade both at the mRNA as well as the protein level. Our study demonstrates that ATF3 mediated TGRL lipolysis induced inflammation and apoptosis is dependent on the TGF-β/Smad signaling pathway.




Figure 4. TGF-β receptor inhibitor (ALK) significantly suppressed TGRL lipolysis induced ATF3 expression. HAEC were exposed to T, TL or 20 ng/mL human TGF-β1 or TL+ALK for 3 h. A) mRNA expression. N=3, P≤0.05. *=TL compare to T, #=TL+ALK compared to TL. B) Protein expression. N=3, P≤0.05. *=TL compare to T, #=TL+ALK compared to TL. TL+CD36 antibody (positive/negative control). C) Immunofluorescence images showing ATF3 nucleus accumulation. N=3 coverslips/treatment group, Bar=20 μm.


Importance of the study: Our findings demonstrate novel interactions between an important extracellular cytokine (TGF-β) and a nuclear transcription factor (ATF3) thought to be central to regulating endothelial inflammatory responses associated with lipid induced vascular injury.


Acknowledgements: This study was supported by grants from NIH-HL55667 and NIA-AG039094, and Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research Fund.



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