Rupture of fetal membranes (ROM) can initiate parturition at both term and preterm. Collagen III in the compact layer of the amnion contributes to the tensile strength of fetal membranes. However, the upstream signals triggering collagen III degradation remain mostly elusive. In this study, we investigated the role of cortisol regenerated by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in collagen III degradation in human amnion fibroblasts with an aim to seek novel targets for the prevention of preterm premature ROM (pPROM)-elicited preterm birth. Human amnion tissue and cultured amnion tissue explants and amnion fibroblasts were used to study the regulation of collagen III, which is composed of three identical 3α 1 chains (COL3A1), by cortisol. Cortisol decreased COL3A1 protein but not mRNA abundance in a concentration-dependent manner. Cortisone also decreased COL3A1 protein, which was blocked by 11β-HSD1 inhibition. The reduction in COL3A1 protein by cortisol was not affected by a transcription inhibitor but was further enhanced by a translation inhibitor. Autophagic pathway inhibitor chloroquine or siRNA-mediated knock-down of ATG7, an essential protein for autophagy, failed to block cortisol-induced reduction in COL3A1 protein abundance, whereas proteasome pathway inhibitors MG132 and bortezomib significantly attenuated cortisol-induced reduction in COL3A1 protein abundance. Moreover, cortisol increased COL3A1 ubiquitination and the reduction of COL3A1 protein by cortisol was blocked by PYR-41, a ubiquitin-activating enzyme inhibitor. Conclusively, cortisol regenerated in amnion fibroblasts may be associated with ROM at parturition by reducing collagen III protein abundance through a ubiquitin-proteasome pathway.
Yabing Mi, Wangsheng Wang, Jiangwen Lu, Chuyue Zhang, Yawei Wang, Hao Ying and Kang Sun
Seung-Chul Kim, Jae-Eon Lee, Seong Soo Kang, Hoe-Saeng Yang, Sun Suk Kim and Beum-Soo An
Oxytocin (OXT) is a peptide hormone that plays a central role in the regulation of parturition and lactation. OXT signaling is mediated by OXT receptor (OXTR), which shows species- and tissue-specific expressions and gene regulation. In the present study, we examined the synthesis of OXT and OXTR in human placenta tissue according to gestational age. A total of 48 placentas were divided into early preterm, late preterm and term groups depending on gestational age, and expression of OXT and OXTR was evaluated. First, OXT and OXTR mRNA and protein were detected in normal placenta tissue via Q-PCR, Dot-blot and Western blot assay. Both OXT and OXTR levels in normal placenta increased gradually in the late stage of pregnancy, suggesting that local OXT may play a critical role in the function of the placenta. To determine the regulatory mechanism of OXT, placental BeWo cells were administrated estrogen (E2) or progesterone (P4), and expression of OXT and OXTR was tested. The mRNA and protein levels of OXT and OXTR were upregulated by E2 but blocked by co-treatment with P4. In order to confirm the estrogen receptor (ESR)-mediated signaling, we administrated ESR antagonists together with E2 to BeWo cells. As a result, both OXT and OXTR were significantly altered by ESR1 antagonist (MPP) while moderately regulated by ESR2 antagonist (PHTPP). These results suggest that OXT and OXTR are controlled mainly by E2 in the placenta via ESR1 and thus may play physiological functions in the human placenta during the late stage of pregnancy.
Yi Lu, Wang-sheng Wang, Yi-kai Lin, Jiang-wen Lu, Wen-jiao Li, Chu-yue Zhang and Kang Sun
Our previous studies have demonstrated that human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein of inflammation, wherein SAA1 may participate in parturition by inducing a number of inflammation mediators including interleukine-1β, interleukine-6 and prostaglandin E2. However, the regulation of SAA1 expression in the fetal membranes remains largely unknown. In the current study, we examined the regulation of SAA1 expression by cortisol, a crucial steroid produced locally in the fetal membranes at parturition, and the interaction between cortisol and SAA1 in the feed-forward induction of SAA1 expression in human amnion fibroblasts. Results showed that cortisol-induced SAA1 expression in a concentration-dependent manner, which was greatly enhanced by SAA1 despite modest induction of SAA1 expression by itself. Mechanism studies revealed that the induction of SAA1 expression by cortisol and SAA1 was blocked by either the transcription factor STAT3 antagonist AZD0530 or siRNA-mediated knockdown of STAT3. Furthermore, cortisol- and SAA1-induced STAT3 phosphorylation in a sequential order with the induction by SAA1 preceding the induction by cortisol. However, combination of cortisol and SAA1 failed to further intensify the phosphorylation of STAT3. Consistently, cortisol and SAA1 increased the enrichment of STAT3 at the SAA1 promoter. Taking together, this study has demonstrated that cortisol and SAA1 can reinforce each other in the induction of SAA1 expression through sequential phosphorylation of STAT3. The enhancement of cortisol-induced SAA1 expression by SAA1 may lead to excessive SAA1 accumulation resulting in parturition-associated inflammation in the fetal membranes.
Young Sun Kang, Yun Gyu Park, Bo Kyung Kim, Sang Youb Han, Yi Hwa Jee, Kum Hyun Han, Mi Hwa Lee, Hye Kyoung Song, Dae Ryong Cha, Shin Wook Kang and Dae Suk Han
Angiotensin II (Ang-II) and vascular endothelial growth factor (VEGF) have an important role in the pathogenesis of diabetic nephropathy, but the signaling cascade of VEGF regulation in response to Ang-II in podocytes is largely unknown. In these experiments, we looked at the effect of Ang-II on the production of VEGF, and investigated whether VEGF production depends on the p38 mitogen activated protein kinase (MAPK) pathway in cultured mouse podocytes. Incubation of podocytes with Ang-II induced a rapid increase in VEGF mRNA expression and protein synthesis as well as its transcriptional activity in an Ang-II dose-dependent manner. To further define the role of angiotensin type 1 (AT1) and type 2 (AT2) receptors involved in Ang-II-mediated VEGF synthesis, the effects of selective AT1 and AT2 receptor antagonists were evaluated. Prior treatment with losartan significantly inhibited VEGF mRNA and protein synthesis induced by Ang-II, which suggests that the AT1 receptor is involved in Ang-II-mediated VEGF synthesis. Furthermore, stimulation of the cells with Ang-II increased both phosphorylation of p38 MAPK and MAP kinase kinase 3/6 (MKK3/6). Additionally, Ang-II enhanced the DNA binding activity to cAMP response element binding protein (CREB) and phosphorylation of CREB. In addition, to investigate the role of p38 MAPK in Ang-II-induced VEGF synthesis, podocytes were pretreated with or without the p38 MAPK inhibitor, SB203580 for 24 h to observe whether Ang-II-mediated VEGF synthesis was inhibited by blocking p38 MAPK. The addition of SB203580 led to a marked inhibition of the increased VEGF mRNA and protein production induced by Ang-II in a dose-dependent manner. Taken together, these results suggest that Ang-II stimulates the synthesis of VEGF in podocytes and the production of VEGF induced by Ang-II is mediated, in part, through the activation of the p38 MAPK pathway.
Huixia Li, Zhuanmin Zhang, Dongxu Feng, Lin Xu, Fang Li, Jiali Liu, Xinxin Jin, Zhuang Qian, Xiaomin Kang and Hongzhi Sun
Progranulin (PGRN), a multifunctional protein implicated in embryonic development and immune response, was recently introduced as a novel marker of chronic inﬂammation related with insulin resistance in obesity and type 2 diabetes mellitus. However, the potential mechanisms of PGRN on insulin signaling pathways are poorly understood. In this study, PGRN mediated the chemotaxis of RAW264.7, impaired insulin action and stimulated production of inflammatory factors in adipocytes, which was accompanied by increased c-Jun N-terminal kinase (JNK) activation and serine phosphorylation of insulin receptor substrate-1. PGRN knockdown partially led to an increase in insulin action as well as a decrease in the JNK activation and extracellular signal-regulated kinase phosphorylation in cells exposed to tumor-necrosis factor-α (TNF-α). Meanwhile, PGRN treatment resulted in an elevation of transcription factor nuclear factor κB (NF-κB) nuclear translocation and acetylation, and increased Il-1b, Il6, Tnf-a expression, whereas NF-κB inhibition reversed PGRN-induced insulin action impairment and inflammatory gene expression. Finally, we showed that sirtuin 1 (SIRT1) expression was downregulated by PGRN treatment, whereas SIRT1 overexpression improved PGRN-induced insulin resistance, NF-κB activation, and inflammatory gene expression. Our results suggest that PGRN regulates adipose tissue inflammation possibly by controlling the gain of proinflammatory transcription in a SIRT1-NF-κB dependent manner in response to inducers such as fatty acids and endoplasmic reticulum stress.