Kreisberg J I, Garoni J A, Radnik R, Ayo S H
Audie Murphy Veteran's Administration Hospital, San Antonio, Texas.
Kidney Int. 1994 Oct;46(4):1019-24. doi: 10.1038/ki.1994.362.
Previously, we reported that mesangial cells increased fibronectin, laminin and type IV collagen synthesis when cultured in the presence of high glucose (30 mM). Although mRNA levels for all three extracellular matrix (ECM) proteins were also increased in high glucose conditions, the mechanism for this increase was not known. In order to determine whether increased transcription was involved in the observed increase in fibronectin mRNA levels mesangial cells were transfected with a construct containing the 5'-flanking region of the fibronectin (FN) gene [position +69 to -510 base pairs (bp)] fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene [FN-CAT (-510)]. Cells were transiently and stably transfected with this construct. Under serum-free conditions, high glucose increased CAT activity only in the presence of TGF beta 1 (referred to as TGF beta). The experiments were performed without serum because FN-CAT (-510) contains a serum responsive element. The increase in CAT was approximately twofold in transiently transfected cells and threefold in stably transfected cells. TGF beta alone increased CAT activity approximately 30%. Stimulation of fibronectin gene expression appeared to occur at the level of a cAMP response element (CRE) located -170 bp of the FN gene because cells transfected with a construct containing an oligonucleotide encoding for this CRE fused to a minimal fibronectin promoter (-56 bp) and a CAT reporter gene [CRE (-170) FN-CAT] displayed similar increments of CAT activity after treatment with high glucose and TGF beta. Gel shift mobility assays with a CRE oligonucleotide revealed multiple complexes with mesangial cell nuclear proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
