Fluid secretion provides a critical distending pressure necessary for fetal lung growth and differentiation. This laboratory first demonstrated that ClC-2, like CFTR, is highly expressed in rabbit fetal airway epithelia and even more rapidly down regulated. The goal of this proposal is to elucidate the mechanism by which the ClC-2 fetal lung chloride channel is highly expressed in fetal airway epithelia and then down-regulated in lung at birth, and to compare this regulation with that of the CFTR. The hypothesis is that ClC-2 gene expression is controlled by perinatal regulation of specific nuclear transcription factors. We have preliminary data indicating a key role for the SP-1 family and the TTF-1 (thyroid transcription factor).
Aim 1 is to identify the ClC-2 promoter elements that drive or inhibit gene expression using a combination of promoter-luciferase assays, DNA foot printing, and mutation analyses.
Aim 2 is to study the perinatal regulation of ClC-2 by SP-1 (and other transcription factors). Two important regulators of SP-1 activity, phosphorylation/dephosphorylation and butyrate stimulation of expression, will be a focus of these studies.
Aim 3 is to study ClC-2 dependent chloride transport under conditions designed to upregulate or down-regulate ClC-2 gene expression. In this section, we will utilize Ussing Chamber models of polarized distal rat lung epithelial cells treated to upregulate SP-I/SP-3 or TTF-I. This refocused proposal should provide important new information with regard to perinatal chloride channel regulation in the lung and fuel thinking into the design of future therapies for pulmonary hypoplasia, cystic fibrosis, and related airway diseases thought to result from inadequate airway hydration or fluid pressure.
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