Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression.
Chronic exposure of HIT-T15 beta cells to elevated glucose concentrations leads to decreased insulin gene transcription. The reduction in expression is accompanied by diminished binding of a glucose-sensitive transcription factor (termed GSTF) that interacts with two (A+T)-rich elements within the 5' flanking control region of the insulin gene. In this study we examined whether GSTF corresponds to the recently cloned insulin gene transcription factor STF-1, a homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and pancreas. We found that an affinity-purified antibody recognizing STF-1 supershifted the GSTF activator complex formed from HIT-T15 extracts. In addition, we demonstrated a reduction in STF-1 mRNA and protein levels that closely correlated with the change in GSTF binding in HIT-T15 cells chronically cultured under supraphysiologic glucose concentrations. The reduction in STF-1 expression in these cells could be accounted for by a change in the rate of STF-1 gene transcription, suggesting a posttranscriptional control mechanism. In support of this hypothesis, no STF-1 mRNA accumulated in HIT-T15 cells passaged in 11.1 mM glucose. The only RNA species detected was a 6.4-kb STF-1 RNA species that hybridized with 5' and 3' STF-1-specific cDNA probes. We suggest that the 6.4-kb RNA represents an STF-1 mRNA precursor and that splicing of this RNA is defective in these cells. Overall, this study suggests that reduced expression of a key transcriptional regulatory factor, STF-1, contributes to the decrease in insulin gene transcription in HIT-T15 cells chronically cultured in supraphysiologic glucose concentration.
Olson
L K
LK
Department of Medicine, University of Minnesota, Minneapolis 55455, USA.
Sharma
A
A
Peshavaria
M
M
Wright
C V
CV
Towle
H C
HC
Rodertson
R P
RP
Stein
R
R
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D015870:Gene Expression / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics*; D007515:Islets of Langerhans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D015534:Trans-Activators / Q000096:biosynthesis* / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects
D017931:DNA Primers; D018398:Homeodomain Proteins; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C084010:pancreatic and duodenal homeobox 1 protein; D015500:Chloramphenicol O-Acetyltransferase; D005947:Glucose