Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance.
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.
Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai, India.
Debarchana
D
Saha
Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai, India.
Swanand
S
Koli
Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai, India.
Mandar
M
Patgaonkar
Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai, India.
Kudumula Venkata Rami
KV
Reddy
D001665:Binding Sites; D015415:Biomarkers; D002460:Cell Line; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D009092:Mucous Membrane / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014621:Vagina / Q000378:metabolism*; D055542:alpha-Globins / Q000235:genetics*; D055544:beta-Globins / Q000235:genetics*
C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D015415:Biomarkers; D016207:Cytokines; D016328:NF-kappa B; D009570:Nitriles; D012333:RNA, Messenger; D013450:Sulfones; D014157:Transcription Factors; D055542:alpha-Globins; D055544:beta-Globins