Expression Of Hemoglobin-Alpha And Beta Subunits In Human Vaginal Epithelial Cells And Their Functional Significance

Hemoglobin (Hb) is a major protein involved in transport of oxygen (O-2). It consists of Hb-alpha and Hb-beta 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-alpha and Hb-beta proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-alpha and Hb-beta proteins (4) to decipher the significance of the Hb-alpha and Hb-beta expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-alpha 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-alpha and Hb-beta at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10 mu g/m1 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-kappa B 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 (5 mu M for 24 hrs) could neutralize the effect of LPS-induced p65-NF-kappa B activity and represses the production'of Hb-alpha and Hb-beta. The results of EMSA revealed the presence of putative binding sites of NF-kappa B in the human Hb-alpha promoter region (nt-115 to-106). ChIP analysis confirmed the binding of NF-kappa B to Hb-alpha promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-alpha and Hb-beta and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-KB regulatory sequences in Hb-alpha promoter, whose activation is associated with immune response of hPVECs, indicating Hb-alpha and Hb-beta may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.