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Expression of hemoglobin-? and ? subunits in human vaginal epithelial cells and their functional significance.

ABSTRACT: Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-? and Hb-? 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-? and Hb-? proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-? and Hb-? proteins (4) to decipher the significance of the Hb-? and Hb-? expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-? 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-? and Hb-? at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10?g/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-?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?M for 24 hrs) could neutralize the effect of LPS-induced p65-NF-?B activity and represses the production`of Hb-? and Hb-?. The results of EMSA revealed the presence of putative binding sites of NF-?B in the human Hb-? promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-?B to Hb-? promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-? and Hb-? and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-?B regulatory sequences in Hb-? promoter, whose activation is associated with immune response of hPVECs, indicating Hb-? and Hb-? may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.

SUBMITTER: Saha D

PROVIDER: S-EPMC5298339 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance.

Saha Debarchana D Koli Swanand S Patgaonkar Mandar M Reddy Kudumula Venkata Rami KV

PloS one 20170208 2

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 ...[more]

PMID: 28178273

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Project description:The opportunistic pathogen Candida glabrata is the second most frequent causative agent of vulvovaginal candidiasis (VVC), a disease that affects 70-75% of women at least once during their life. However, C. glabrata is almost avirulent in mice and normally incapable of inflicting damage to vaginal epithelial cells in vitro. We thus proposed that host factors present in vivo may influence C. glabrata pathogenicity. We, therefore, analyzed the impact of albumin, one of the most abundant proteins of the vaginal fluid. The presence of human, but not murine, albumin dramatically increased the potential of C. glabrata to damage vaginal epithelial cells. This effect depended on macropinocytosis-mediated epithelial uptake of albumin and subsequent proteolytic processing. The enhanced pathogenicity of C. glabrata can be explained by a combination of beneficial effects for the fungus, which includes an increased access to iron, accelerated growth, and increased adhesion. Screening of C. glabrata deletion mutants revealed that Hap5, a key regulator of iron homeostasis, is essential for the albumin-augmented damage potential. The albumin-augmented pathogenicity was reversed by the addition of iron chelators and a similar increase in pathogenicity was shown by increasing the iron availability, confirming a key role of iron. Accelerated growth not only led to higher cell numbers, but also to increased fungal metabolic activity and oxidative stress resistance. Finally, the albumin-driven enhanced damage potential was associated with the expression of distinct C. glabrata virulence genes. Transcriptional responses of the epithelial cells suggested an unfolded protein response (UPR) and ER-stress responses combined with glucose starvation induced by fast growing C. glabrata cells as potential mechanisms by which cytotoxicity is mediated.Collectively, we demonstrate that albumin augments the pathogenic potential of C. glabrata during interaction with vaginal epithelial cells. This suggests a role for albumin as a key player in the pathogenesis of VVC.

Project description:PURPOSE: The family of natriuretic peptides (NPs); atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as three associated receptors (NPRs); natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B), and natriuretic peptide receptor C (NPR-C) has never been documented in human lens epithelial cells. The study described herein was designed to demonstrate both expression and functionality of components of the natriuretic peptides and natriuretic peptide receptors in the human lens epithelial cell line, HLE-B3 and in normal human lens epithelial cell cultures (nHLE). METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) along with confirmation by DNA sequencing and real-time quantitative RT-PCR was used to identify and demonstrate expression of mRNA for the natriuretic peptide family. Authentication of protein expression of the natriuretic peptide receptors was determined by using formaldehyde-fixed, Saponin-permeabilized cells (HLE-B3) or methanol:acetone-fixed and permeabilized cells (nHLE) using conventional immunofluorescence techniques. Enzyme-linked immunosorbent assay was used to determine cyclic GMP (cGMP) activity as stimulated by exogenous addition of natriuretic peptides. RESULTS: Using RT-PCR with confirmation by DNA sequencing and real-time quantitative RT-PCR, HLE-B3 cells were shown to express mRNA for ANP, BNP, and CNP along with their associated receptors. Conventional immunofluorescence on the permeabilized cells confirmed positive diffuse staining indicating the presence of the three natriuretic peptide receptors in both HLE-B3 and nHLE cells. All three natriuretic peptides educe a cGMP response in the rank order CNP>>ANP approximately BNP indicating that the natriuretic peptide family is functional in HLE-B3 cells. CONCLUSIONS: The data indicates that ANP, BNP, and CNP and natriuretic peptide receptor transcripts are expressed and are functional in human lens epithelial cells. The cellular expression of NPs and NPRs, as well as the demonstration that all three NPs activate guanylyl cyclase suggests a potential role in maintaining lens epithelial cell homeostasis.

Dataset Information

Expression of hemoglobin-? and ? subunits in human vaginal epithelial cells and their functional significance.

Publications

Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance.

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