Saha Debarchana, Koli Swanand, Patgaonkar Mandar, Reddy Kudumula Venkata Rami
Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai, India.
PLoS One. 2017 Feb 8;12(2):e0171084. doi: 10.1371/journal.pone.0171084. eCollection 2017.
Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11-7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-κB to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-β and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-κB regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-β may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.
血红蛋白（Hb）是参与氧气（O2）运输的主要蛋白质。它由Hb-α和Hb-β亚基组成，通常由红系谱系细胞表达。然而，直到最近，尚不清楚阴道细胞等非红系细胞是否合成Hb以及它是否具有任何功能意义。因此，我们设计了以下目标：（1）建立人原代阴道上皮细胞（hPVECs）的体外培养系统，（2）确定hPVECs是否真的合成Hb-α和Hb-β蛋白，（3）评估脂多糖（LPS）对Hb-α和Hb-β蛋白表达的影响，（4）解读hPVECs中Hb-α和Hb-β表达的意义，以及（5）确定调节hPVECs中Hb-α表达的分子机制。为完成这些研究，我们应用了一系列检测方法，如逆转录聚合酶链反应（RT-PCR）、实时定量逆转录聚合酶链反应（qRT-PCR）、流式细胞术、蛋白质免疫印迹法和免疫荧光法、电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）。结果显示hPVECs中Hb-α和Hb-β在mRNA和蛋白质水平均有表达。LPS处理（10μg/ml，6小时）后表达显著上调，这些结果与LPS在人阴道上皮细胞系（VK2/E6E7）中诱导的表达相当。这些细胞组成性地产生低水平的促炎细胞因子（IL-6）和抗炎细胞因子（IL-10）。此外，在hPVECs和VK2/E6E7细胞中，磷酸化（p65）-核因子κB（NF-κB）对LPS的反应随着IL-6、Toll样受体4（TLR4）和人β-防御素1（hBD-1）表达的增加而上调。然而，Bay 11-7082处理（5μM，24小时）可中和LPS诱导的p65-NF-κB活性的作用，并抑制Hb-α和Hb-β的产生。EMSA结果显示人Hb-α启动子区域（nt-115至-106）存在NF-κB的假定结合位点。ChIP分析证实NF-κB与Hb-α启动子结合。总之，本研究结果首次揭示hPVECs合成Hb-α和Hb-β，且其表达与VK2/E6E7细胞的表达相当。在Hb-α启动子中鉴定出NF-κB调节序列，其激活与hPVECs的免疫反应相关，表明Hb-α和Hb-β可能作为针对阴道炎症/感染的内源性抗菌防御蛋白。
