ABSTRACT
We report the cDNA cloning and characterization of ZNF236, a novel Kruppel-like zinc-finger gene initially identified by its glucose-regulated expression in human mesangial cells using mRNA differential display. Using the differential display fragment as a probe, we screened a human fetal kidney cDNA library and isolated several clones representing two differently spliced mRNA transcripts, designated ZNF236a and -b. Both transcripts were identical apart from the presence of an additional exon in ZNF236a that truncates the open reading frame. RT-PCR analysis confirmed the expression of both transcripts to be upregulated in human mesangial cells in response to elevated levels of d-glucose. ZNF236a and -b cDNAs encode polypeptides of 174 and 204 kDa, containing 25 and 30 C(2)H(2) zinc-finger motifs, respectively. Northern blot analysis showed that ZNF236 is ubiquitously expressed in all human tissues tested. Expression levels were highest in skeletal muscle and brain, intermediate in heart, pancreas, and placenta, and lowest in kidney, liver, and lung. Southern zoo blot analysis indicated that ZNF236 is conserved in the genomes of all mammalian species tested, but not in yeast. The mapping of ZNF236 to human chromosome 18q22-q23, close to the IDDM6 locus, coupled with the glucose-regulated expression of the gene in human mesangial cells, suggests that ZNF236 may be a candidate gene for diabetic nephropathy.
Subject(s)
Carrier Proteins/genetics , Chromosomes, Human, Pair 18/genetics , DNA-Binding Proteins/genetics , Repressor Proteins , Transcription Factors/genetics , Zinc Fingers/genetics , Adult , Alternative Splicing , Amino Acid Sequence , Animals , Blotting, Northern , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Female , Glucose/physiology , Humans , In Situ Hybridization, Fluorescence , Kruppel-Like Transcription Factors , Molecular Sequence Data , Protein Isoforms/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
Diabetic nephropathy is characterised by an accumulation of extracellular matrix proteins in the glomerular mesangium. Hyperglycaemia is a major factor promoting this progressive expansion of the mesangial matrix. We have used the technique of mRNA differential display to investigate changes in gene expression in cultured human mesangial cells following long-term (21 days) exposure to either physiologic (4 mM) or pathologic (30 mM) D-glucose concentrations. Approximately 12,000 mRNA species were screened for evidence of altered expression and several hundred candidate cDNA fragments were obtained. Northern blot and RT-PCR analysis of ten randomly chosen candidate cDNA fragments revealed three exhibiting increased mRNA expression under elevated D-glucose levels. Nucleotide sequence analysis identified two of the cDNA fragments as encoding prolyl 4-hydroxylase alpha-subunit and thrombospondin-1. The third cDNA fragment represents a novel glucose-regulated gene, encoding a putative zinc finger protein. Upregulated expression of these genes in response to high levels of D-glucose may contribute significantly to the disease process. mRNA differential display is a useful tool to investigate the mechanism of diabetic nephropathy.
