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LPS-TLR4 signaling attenuates CHOP-mediated apoptosis under endoplasmic reticulum stress conditions during porcine
Gyu-Hyun Lee1, Cheng-Lin Zhan1, Song-Hee Lee1
1Department of Animal Science, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
Introduction:
Persistent endoplasmic reticulum (ER) stress impairs early embryonic development by inducing apoptosis through C/EBP homologous protein (CHOP). Toll-like receptor 4 (TLR4), traditionally recognized for its role in innate immunity, has recently emerged as a modulator of intracellular stress responses. Lipopolysaccharide (LPS), a natural TLR4 agonist derived from Gram-negative bacteria, elicits both pro-inflammatory and cytoprotective effects depending on the cellular context and dosage. This study aimed to elucidate the role of TLR4 signaling in the regulation of CHOP-mediated apoptosis during porcine preimplantation development under ER stress.
Methods:
Porcine embryos were treated with tunicamycin (TM, 5 nM) to induce ER stress and co-treated with LPS (10 μM) to activate TLR4 signaling. Developmental competence was assessed by blastocyst formation rates, total cell number, and markers of apoptosis and autophagy.
Results:
LPS treatment significantly improved blastocyst formation rates compared to TM groups (TM: 37.50 ± 4.77% vs. TM+LPS: 52.89 ± 4.86%). Consistent with this improvement, the total cell number per blastocyst was significantly restored by LPS co-treatment (Control: 55.63 ± 2.15 vs. TM: 38.61 ± 2.57; TM+LPS: 48.84 ± 0.83), confirming enhanced cell proliferation under ER stress conditions. LPS co-treatment markedly reduced CHOP protein expression and suppressed ATF4 expression, indicating alleviation of PERK-ATF4-CHOP signaling. Additionally, autophagy and apoptosis were attenuated, as evidenced by a significantly decreased LC3-II/LC3-I ratio and a reduced number of TUNEL-positive cells. Notably, TLR4 knockdown abolished these LPS-mediated protective effects, confirming the requirement of TLR4 in mitigating ER stress-induced damage.
Conclusion:
These findings demonstrated that LPS-mediated TLR4 signaling suppressed CHOP-induced apoptosis and autophagy under persistent ER stress, thereby improving embryonic viability. This study provides novel mechanistic insights into the non-canonical role of TLR4 in early embryonic development and highlights its therapeutic potential for improving in vitro embryo culture systems.
Insights
Toll-like receptor 4 (TLR4) signaling, activated by lipopolysaccharide (LPS), mitigates endoplasmic reticulum (ER) stress-induced apoptosis in porcine embryos. This improves early embryonic development and offers potential for enhanced in vitro embryo culture.
Area of Science:
- Reproductive Biology
- Cellular Stress Response
- Immunology
Background:
- Persistent endoplasmic reticulum (ER) stress, marked by C/EBP homologous protein (CHOP) induction, detrimentally affects early embryonic development via apoptosis.
- Toll-like receptor 4 (TLR4), known for immune functions, also modulates intracellular stress responses.
- Lipopolysaccharide (LPS), a TLR4 agonist, exhibits context-dependent effects, including cytoprotection.
Purpose of the Study:
- To investigate the role of TLR4 signaling in regulating CHOP-mediated apoptosis during porcine preimplantation development under ER stress.
- To determine if LPS can counteract ER stress-induced developmental impairment in porcine embryos.
Main Methods:
- Porcine embryos were subjected to tunicamycin (TM) to induce ER stress, with concurrent lipopolysaccharide (LPS) treatment to activate Toll-like receptor 4 (TLR4) signaling.
- Developmental competence was evaluated by blastocyst formation rates, total cell counts, and assessments of apoptosis and autophagy markers.
Main Results:
- LPS co-treatment significantly enhanced blastocyst formation rates and restored total cell numbers in TM-treated porcine embryos.
- LPS administration reduced CHOP and ATF4 expression, indicating suppression of the PERK-ATF4-CHOP pathway.
- Autophagy and apoptosis markers (LC3-II/LC3-I ratio, TUNEL-positive cells) were significantly attenuated by LPS treatment.
- TLR4 knockdown negated the protective effects of LPS, confirming TLR4's essential role.
Conclusions:
- LPS-mediated TLR4 signaling effectively suppresses CHOP-induced apoptosis and autophagy under persistent ER stress, thereby improving porcine embryonic viability.
- This study reveals a non-canonical role for TLR4 in early embryonic development, suggesting its therapeutic potential for improving in vitro embryo culture systems.
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