Abstract
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.
Highlights
Vaginal epithelium of the female reproductive tract (FRT) is constantly exposed to a large number of pathogens, which cause sexually transmitted infections (STIs) [1, 2]
Our results demonstrated that Hb-α and Hb-β proteins were constitutively expressed by human primary vaginal epithelial cells (hPVECs) and are comparable with the expression observed in VK2/E6E7 cell line
The results indicated that hPVECs and VK2/E6E7 cells did not stain for vimentin, suggesting the cultures were devoid of stromal cells (Fig 4a–4f)
Summary
Vaginal epithelium of the female reproductive tract (FRT) is constantly exposed to a large number of pathogens, which cause sexually transmitted infections (STIs) [1, 2]. Vaginal epithelial cells (VECs) of FRT play an important role in initiation, regulation and resolution of both innate and adaptive immune functions [3, 4]. The Hb-α gene is embedded in an open chromatin conformation, whereas β-globin gene cluster is packaged into an inactive heterochromatin in all the cell types [9]. It is not known why Hb-α and Hb-β genes are regulated so differently in vaginal epithelial cells even though when their expression is always tightly linked
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