Proteomic Analysis of Human
Bladder Epithelial Cells
by 2D Blue Native SDS-PAGE Reveals TCDD-Induced Alterations of Calcium
and Iron Homeostasis Possibly Mediated by Nitric Oxide
Posted on 2015-01-02 - 00:00
A proteomic analysis of the interaction
among multiprotein complexes
involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated
toxicity in urinary bladder epithelial RT4 cells was performed using
two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the
protein complexes, unexposed and TCDD-exposed cells were fractionated.
BN/SDS-PAGE of the resulting fractions led to an effective separation
of proteins and protein complexes of various origins, including cell
membrane, mitochondria, and other intracellular compartments. Major
differences between the proteome of control and exposed cells involved
the alteration of many calcium-regulated proteins (calmodulin, protein
S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated
proteins (ferritin, heme-binding protein 2, transferrin). On the basis
of these findings, the intracellular calcium concentration was determined,
revealing a significant increase after 24 h of exposure to TCDD. Moreover,
the concentration of the labile iron pool (LIP) was also significantly
elevated in TCDD-exposed cells. This increase was strongly inhibited
by the calmodulin (CaM) antagonist W-7, which pointed toward a possible
interaction between iron and calcium signaling. Because nitric oxide
(NO) production was significantly enhanced in TCDD-exposed cells and
was also inhibited by W-7, we hypothesize that alterations in calcium
and iron homeostasis upon exposure to TCDD may be linked through NO
generated by CaM-activated nitric oxide synthase. In our model, we
propose that NO produced upon TCDD exposure interacts with the iron
centers of iron-regulatory proteins (IRPs) that modulate the alteration
of ferritin and transferrin, resulting in an augmented cellular LIP
and, hence, increased toxicity.
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Verma, Nisha; Pink, Mario; Petrat, Frank; Rettenmeier, Albert W.; Schmitz-Spanke, Simone (2016). Proteomic Analysis of Human
Bladder Epithelial Cells
by 2D Blue Native SDS-PAGE Reveals TCDD-Induced Alterations of Calcium
and Iron Homeostasis Possibly Mediated by Nitric Oxide. ACS Publications. Collection. https://doi.org/10.1021/pr501051f
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AUTHORS (5)
NV
Nisha Verma
MP
Mario Pink
FP
Frank Petrat
AR
Albert W. Rettenmeier
SS
Simone Schmitz-Spanke