Perinatal regulation of the ClC-2 chloride channel in lung is mediated by Sp1 and Sp3.
Mechanisms responsible for regulation of pulmonary epithelial chloride-channel expression in the perinatal period are under investigation to better understand normal lung development and airway disease pathogenesis. The ClC-2 epithelial chloride channel is regulated by changes in pH and volume and is most abundant in lung during fetal development. In this study, we identify and sequence the ClC-2 promoter, which is GC rich and lacks a TATA box. By construction of a series of promoter-luciferase constructs, a 67-bp GC box-containing sequence in the promoter is shown to be critical to ClC-2 expression in primary and immortalized fetal lung epithelial cells. Electrophoretic mobility shift assays and antibody supershifts demonstrate that the Sp1 and Sp3 transcription factors are expressed in fetal lung nuclei and interact with the GC box sequences in the promoter. Immunoblotting techniques demonstrate that Sp1 and Sp3 are perinatally downregulated in the lung with the same temporal sequence as ClC-2 downregulation. This work suggests that Sp1 and Sp3 activate ClC-2 gene transcription and that reduction in Sp1 and Sp3 at birth explains perinatal downregulation of ClC-2 in the lung.
Chu
S
S
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-2533, USA. shijian@welchlink.welch.jhu.edu
Blaisdell
C J
CJ
Liu
M Z
MZ
Zeitlin
P L
PL
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D000090007:CLC-2 Chloride Channels; D002478:Cells, Cultured; D018118:Chloride Channels / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005314:Embryonic and Fetal Development / Q000502:physiology*; D005333:Fetus; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008168:Lung / Q000196:embryology / Q000254:growth & development / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D008156:Luciferases