TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells - PubMed (original) (raw)

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

Larissa Eiselein et al. PLoS One. 2015.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 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 of 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.

Fig 2. Western Blots of Smad2 phosphorylation at 1.5 h and nuclear expression of Smad4 at 3 h.

A) Smad2 and Phospho-Smad2 protein. Western Blot (a) and densitometry quantification (b) of Lysates from endothelial cells treated with TGRL (150 mg/dL) + LpL (2 U/mL) TL for 1.5 h (TL1.5) show significantly increased phosphorylation of Smad2 compared to cells treated with media (M), TGRL (T), LpL (L) or TL for 0.5 h (TL0.5). Addition of 10 μM of 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) Smad4 nuclear expression comparing M, L, TL and TLA. Cytosolic fractions were also run on the same blot corresponding to the nuclear fraction. Levels of Smad4 were too low to detect when loaded at a proteins concentration equivalent to the level of protein loaded for the nuclear fraction. These lanes were removed for clarity. Addition of 10 μM of ALK, TGF-β receptor inhibitor, suppressed but did not completely abrogate lipolysis induced Smad4 nuclear accumulation.

Fig 3. Smad2 phosphorylation at 1.5 h and Smad4 nucleus accumulation at 3 h by lipolysis products.

A) The nuclear isolated phosphorylated Smad2 was only observed with cellular exposure of lipolysis products (TL) or TGF-β1, not with Media (M), LpL (L) or TGRL (T). B) (a) HAEC monolayers show changes in localization of Smad4 from cytosol to nucleus after treatment with lipolysis products compared to controls treated with M, L or T at 1 h. Treatment with lipolysis products with inhibitor ALK (10 μM) 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 products (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).

Fig 4. p53 protein expression by TGRL lipolysis products was suppressed by TGF-β receptor inhibitor, ALK.

Western Blot (a) and densitometry quantification (b) for p53. p53 expression is significantly increased after 1.5 h of treatment with lipolysis products (TL1.5) compared to control cells (M). Treatment with TGRL (T) or LpL (L) does not alter p53 protein content. N = 3/treatment group, * = _P_≤0.05. Expression declines after 3 h of treatment with lipolysis products (TL3) _P_≤0.1.

Fig 5. TGRL lipolysis activates apoptosis both in vitro and vivo.

A) Lipolysis activates caspase-3 activity in HAEC at 3h. B) Caspase-3 protein expression was blocked by TGF-β receptor inhibitor, ALK. Western blots (a) and densitometry quantification (b) for caspase-3. Treatment with lipolysis products increases expression of the active fragment of caspase-3 at 3 h. Additional treatment with 10 μM of inhibitor ALK (TL3+ALK) abrogated the expression of the active caspase-3 fragment. N = 3/treatment group, _P_≤0.05,* = TL compared to TL + ALK. C) TGRL lipolysis products activate apoptosis in mouse carotid artery. (a) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of mouse carotid artery. (b) Percentage of apoptosis of endothelial cells based on FITC and nuclear staining. TGRL lipolysis (TL) significantly induced apoptosis (65%) compare to control in mouse carotid artery. Positive control: DNAse I treated; negative control: without rTdT enzyme. N = 4 mice/group, _P_≤0.05 as significant, * = T or TL compare to media control group, # = TL compare to T. Original magnification ×60, Bar = 20 μm.

Fig 6. The effect of the ALK 4, 5 and 7 inhibitor or anti TGF-β antibody on the TGRL lipolysis induced ATF3 expression.

HAEC were exposed to TGRL (T), TGRL lipolysis products (TL) or 20 ng/mL human TGF-β1 for 3 h. TGF-β receptor inhibitor, ALK significantly suppressed: A) mRNA expression of ATF3. N = 3, _P_≤0.05. * = TL compare to T, # = TL with 10 μM of inhibitor ALK (TL+ALK) compared to TL. B) Western blot (a) and densitometry quantification (b) for ATF3 protein. N = 3, _P_≤0.05. * = TL compare to T, # = TL+ALK compared to TL. TL+CD36 antibody as control (positive/negative). C) Immunofluorescence images showing nucleus accumulation of ATF3. N = 3 coverslips/treatment group, Bar = 20 μm. D) % Translocation of ATF3. N = 6 coverslips/treatment group, _P_≤0.05, * = TL or TGF-β1 compare to T, # = TL+ALK compare to TL, Bar = 20 μm. anti TGF-β1 antibody (Ab) suppressed: E) mRNA expression of ATF3 was significantly suppressed. N = 3, _P_≤0.05. * = TL compare to M, # = TL+ anti TGF-β1 antibody compared to TL. F) Western blot (a) and densitometry quantification (b) ATF3 protein expression was trend toward suppressed significant. N = 3, _P_≤0.05. * = TL or TL + anti TGF-β1 (TLA) compare to M. Ab = anti TGF-β1 antibody.

Fig 7. The effect of the ALK 4, 5 and 7 inhibitor or anti TGF-β antibody on the TGRL lipolysis activated phosphorylation of c-Jun protein expression.

HAEC were exposed to TGRL (T), TGRL lipolysis products (TL) or TL with 10 μM of inhibitor ALK (TL+ALK) for 3 h. TGF-β receptor inhibitor, ALK significantly suppressed: A) Western blot (a) and densitometry quantification (b) for p-c-Jun protein expression. N = 3, _P_≤0.05. * = TL compare to T, # = TL+ALK compared to TL. B) Immunofluorescence images showing nuclear translocation of p-c-Jun. N = 3, Bar = 20 μm. anti TGF-β1 antibody (Ab) suppressed: C) Western blot (a) and densitometry quantification (b) p-c-Jun protein expression was trend toward suppressed significant. N = 3, _P_≤0.05. * = TL or TL + anti TGF-β1 (TLA) compare to M.

Fig 8. TGF-β receptor inhibitor or anti-TGF-β1 suppressed TGRL lipolysis products-induced pro-inflammatory gene expression.

Treatment with lipolysis products increased pro-inflammatory gene expression at 3 h. Effect of 10 μM of inhibitor ALK (TL+ALK): A) mRNA expression of E-selectin, IL-8 and JunB expression was significantly suppressed by TGF-β receptor inhibitor. N = 3, _P_≤0.05. * = TL compare to T, # = TL+ALK compared to TL. B) mRNA expression of IL-6, NFKBIA/IκBA and NFKB1/NFκB (p50)expression was also significantly suppressed by inhibitor. N = 3, _P_≤0.05. * = TL compare to T, # = TL+ALK compared to TL. Effect of 4 μg/mL of anti TGF-β1 antibody: C) mRNA expression of E-selectin and IL-8 expression was significantly suppressed by anti TGF-β1 but not JunB. N = 3, _P_≤0.05. * = TL compare to M, # = TL + anti TGF-β1 compared to TL.

Fig 9. TGRL lipolysis products activate stress response signaling via TGF-β/SMAD Signaling Pathway.

Lipolysis release TGF-β1 and activate phosphorylation of Smad2 and translocation of Smad4 to nucleus. TG-β1 also activate non-Smad signaling pathways ATF3-JNK transcription factor networks. Both Smad and ATF3 further induced pro-inflammatory cytokines and apoptosis which can be inhibited by TGF-β receptor inhibitor, ALK.

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TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells - PubMed (original) (raw)